The Pennsylvania State University

The Graduate School

Eberly College of Science

ION SPECIFICTY AT THE POLYPEPTIDE BACKBONE:

MOLECULAR MECHANISMS OF THE HOFMEISTER EFFECT

A Dissertation in

Chemistry

by

Kelvin Blaine Rembert II

©2014 Kelvin Blaine Rembert II

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

December 2014

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The dissertation of Kelvin B. Rembert II was reviewed and approved* by the following:

Paul S. Cremer Professor of Chemistry & Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee

Scott A. Showalter Associate Professor of Chemistry

William G. Noid Associate Professor of Chemistry

Craig E. Cameron Professor of Biochemistry and Molecular Biology

Barbara J. Garrison Shapiro Professor of Chemistry Head of Department of Chemistry

*Signatures are on file in the graduate school

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ABSTRACT

Ion Specificity at the Polypeptide Backbone:

Molecular Level Mechanisms of the Hofmeister Effect (December 2014)

Kelvin Blaine Rembert II, B.S., University of West Georgia, Carrollton, Georgia

Chair of Advisory Committee: Dr. Paul S. Cremer

The behavior of biomolecules in aqueous solutions is ion specific and follows the

Hofmeister series. Discovered in 1888, the Hofmeister series is a recurring trend that originally ranked cations and anions by their ability to precipitate proteins from aqueous salt solutions.

Over the course of the 21st century however, the Hofmeister effect was found to extend well beyond protein precipitation studies and occurs in myriad bio-chemical and physico-chemical systems. Today, the field remains an active area of research and has gained the attention of many researchers due to the elusiveness in developing a theoretical framework for predicting ion specific effects in water. Only recently has a unified molecular picture began to emerge.

Moreover, utilizing the proper techniques and model systems is key. In this dissertation, we have employed model protein backbones in a set of salt solution studies using NMR, FTIR and thermodynamic phase transition measurements to elucidate both the molecular level and macroscopic details of the Hofmeister effect.

First, we investigate the interactions of Hofmeister anions with a thermoresponsive polymer, poly(N,N-diethylacrylamide) (PDEA). This amide based polymer lacks an NH moiety in its chemical structure and serves as a model to directly test if anions can bind to amide moieties in the absence of an NH site. The lower critical solution temperature (LCST)

iii of PDEA was measured as a function of concentration for eleven sodium salts in aqueous solutions and followed a direct Hofmeister series for the ability of anions to precipitate the

2- 2- 2 - - - polymer. More strongly hydrated anions (CO3 , SO4 , S2O3 , H2PO4 , F , Cl ) linearly decreased the LCST of the polymer with increasing salt concentration. Weakly hydrated anions

- - - - - (SCN , ClO4 , I , NO3 , Br ) increased the LCST at lower salt concentrations and salted the polymer into solution, but salted the polymer out at higher salt concentrations. Proton NMR was used to probe the mechanism of the salting-in effect and showed apparent binding between weakly hydrated anions (SCN- and I-) and the α-protons of the polymer backbone. Additional experiments performed by FTIR found little change in the amide I band, which is consistent with limited, if any, interactions between the salt ions and the carbonyl moiety of the amide.

These results support a molecular mechanism for ion-specific effects on proteins and model amides that does not specifically require an NH group to interact with the anions for salting-in to occur.

Next, the binding sites of a 600 residue protein polymer, (VPGVG)120, were elucidated using a combination of NMR and LCST measurements. It was found that the salting-in effect of large soft anions such as SCN− and I− is due to direct interactions at glycine residues within the polypeptide backbone via a hybrid binding site that consists of the amide dipole and the adjacent α-carbon. The hydrocarbon groups at these sites bear a slight positive charge, which enhances anion binding without disrupting specific hydrogen bonds to water molecules. The hydrophobic side chains do not contribute significantly to anion binding or the corresponding salting-in behavior of the biopolymer. Cl− binds far more weakly to the amide dipole/α-carbon

2− binding site, while SO4 is repelled from both the backbone and hydrophobic side chains of the polypeptide. The Na+ counterions are also repelled from the polypeptide. The identification

iv of these molecular-level binding sites provides new insight into the mechanisms of peptide−ion interactions. Moreover, the location of the amide, whether in a protein backbone or on a polymer side chain, has no appreciable influence on the ability of weakly hydrated anions to bind.

To further explore the glycine residue binding site interaction with anions, we employed triglycine (GGG) along with two variants of the peptide as model systems. These small peptides allowed for the investigation of the influence of size on ion binding as well as the effect of electrostatic interactions of ions with charged termini. We demonstrated by means of NMR and ATR-FTIR spectroscopy that the Hofmeister series for anions changes from a direct to a reversed series upon uncapping the N-terminus of the peptide. Weakly hydrated anions, such as I- and SCN-, interact with the amide backbone, while strongly hydrated anions

2- like SO4 are repelled from it. In contrast, a reversed order of ion interactions was observed at the positively charged, uncapped N-terminus. This by analogy should also be the case at side chains of positively charged amino acids such as lysine and arginine. Overall, the small GGG peptides displayed over an order of magnitude weaker binding compared to the large ELPs mentioned above. These results demonstrate that the specific chemical and physical properties of peptides and proteins play a fundamental role in ion specific effects and provides a route for exploiting these interactions by tuning protein size and the number of basic amino acid residues on the surface.

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TABLE OF CONTENTS

Page LIST OF FIGURES ……….…………………………………………………………… viii LIST OF TABLES ……….……………………………………………………………. xii ACKNOWLEDGEMENT ...………………………………………………………….. xiii DEDICATION …………….…………………………………………………………... xiv

CHAPTER

I INTRODUCTION …………………………………………………………. 1 Background and Hofmeister Series .…………………...... 1 Model Systems ………………………………………………...... 2 Methods ……………………………………………...... 3

II AN NH MOIETY IS NOT REQUIRED FOR ANION BINDING TO AMIDES .....……………………………………………………………….………….. 11 Introduction ………………………………………………………………... 11 Materials and methods ………………………………………...... 13 Results ………………………………………………………...... 15 Discussion & Conclusion ……………………………………...... 19

III MOLECULAR MECHANISMS OF ANION-SPECIFIC BINDING TO PROTEIN BACKBONES ……………………………….…………………………...... 31 Introduction ………………………………………………………………... 31 Materials and methods …………………………………………………….. 33 Results ………………………………………………………...... 35 Discussion …………………………………………………………………. 40 Conclusion …………………………………………………………………. 42

IV SPECIFIC ION EFFECTS ON TRIGLYCINE: REVERSED HOFMEISTER SERIES AND INFLUENCE OF SIZE ON ION BINDING………………. 59 Introduction ………………………………………………………………... 59 Materials and Methods .……………………………………...... 60 Results ……………………………………………………………………... 61 Discussion and Conclusion ...……………………………...... 65

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CHAPTER Page

V BEYOND HOFMEISTER & FUTURE DIRECTIONS ……………...... 75 Perspective ………………………………………………………………… 75 Exploiting Polar/Nonpolar Hybrid Binding Sites …………………………. 76 Pharmaceutical Formulations ……………………………………………… 78

REFERENCES ……………………………………………………………...... 80

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LIST OF FIGURES

Figure Page

1.1 Ribbon structure of a protein illustrating its unique, complex, three-

dimensional structure…………………………………………………….... 5

1.2 The direct Hofmeister series for cations and anions………………………... 6

1.3 Model systems employed. Thermoresponsive polymers (left) undergo

phase transitions at lower critical solution temperatures (LCST) and serve as suitable protein models for hydrophobic collapse. Oligopeptides (right)

such as triglycine and other analogues are investigated to determine the influence of charged and non-charged end groups, as well as molecular size, on ion binding……………………………………………………...... 7

1.4 A representative light scattering plot as a function of temperature for a thermoresponsive protein or biopolymer measured with an OptiMelt™. The lower critical solution temperature value, LCST, is designated as the onset of the increase in light scattering relative to the baseline…………...... 8

1.5 A representative dark-field microscope snapshot of various samples in a microfluidic chip on the linear temperature gradient platform. The linescan is taken at the interface between the soluble and insoluble polymer phases (i.e. the transition temperature) and the right figure corresponds to the scattering intensity as a function of pixel position (i.e. temperature)………. 9

1.6 Multi-spectra display of a representative proton’s chemical shift in the presence of varying salt concentration……………………………………... 10

2.1 Monomeric structures of Poly(N-isopropylacrylamide), PNIPAM, and Poly(N,N-diethylacrylamide), PDEA……………………………………… 22

2.2 (A) LCSTs of PDEA as function of salt concentration for eleven sodium salts. Data were best fit linear or to an expression containing both a linear and nonlinear binding term (see eqn. 1). (B) Residual LCSTs of PDEA versus 6 salts after subtraction of the linear component. The data for NaSCN, NaI, NaClO4, NaNO3, and NaBr were best fit to a binding isotherm. The data points for NaCl are connected with lines as a guide to the eye……………………………………………………………………... 23

2.3 Relative chemical shift changes (∆δ) of N-CH2, Alpha, and Methyl protons of PDEA as a function of salt concentration for (A) Na2SO4, (B) NaCl, (C) NaI, and (D) NaSCN. Data were best fit linear or to an expression containing both a linear and nonlinear binding term (see eqn. 2). Structure of PDEA monomer with circles adjacent indicating their corresponding protons on graphs………………………………………………………...... 24 viii

Figure Page

2.4 Residual change after subtraction of the linear portion in the chemical shift changes for backbone α-protons, left y-axis (black), and residual LCST changes, right y-axis (blue), both as a function of either NaI or NaSCN concentration………………………………………………………………. 25

2.5 FTIR spectrum of PDEA in D2O at 5°C, with individual Gaussian peaks fits (green plots) and cumulative peak fit (red plot)………………………… 27

2.6 The individual amide I peak positions plotted as a function of employed salt concentrations for (A) 1599 cm-1 and (B) 1619 cm-1 peak positions. The yellow highlighted region represents the 2 cm-1 resolution of the spectrometer centered at the amide I band in D2O in the absence of salt…… 28

2.7 Correlation between the salting-out coefficients, c, from data fits to eqn.1 as a function of the hydration entropy of the eleven Hofmeister anions used in this study. The red line is a linear fit for values corresponding to the strongly hydrated anions. The salting out coefficients for strongly hydrated anions show a general dependence on the hydration thermodynamics of the anions while the weakly hydrated anions do not…...... 29

2.8 Correlation between the salting-out coefficients, c, from data fits to eqn.1 as a function of the surface tension increments for the eleven Hofmeister anions used in this study. The green and red lines are both linear fits to the strongly hydrated and weakly hydrated anion values, respectively. The salting out coefficients for both sets of anions show a general dependence on the air/water interface surface tension increments……………………… 30

3.1 Schematic diagram depicting the molecular-level binding sites for SCN−

with (VPGVG)120. As shown, the anion binds preferentially to the Glycine methylene moiety within the amide backbone, but not appreciably to a purely hydrophobic methyl group such as the ones on valine. The anion also binds to some extent with the NH proton. It should be noted that only one resonance structure of SCN− is depicted. The other one consists of two double bonds and the placement of the formal charge on the nitrogen atom.. 44

3.2 Change in LCST (∆LCST) of (VPGVG)120 as a function of salt o concentration for Na2SO4, NaCl, and NaSCN. ∆LCST=0, 31.9 C, corresponds to the LCST of the polymer in the absence of salt…………….. 45

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Figure Page

o 3.3 Proton NMR spectrum showing assignments of (VPGVG)120 at 5 C in a salt-free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide above…………………………………………… 46

1 o 3.4 H NMR NOESY spectrum showing assignments of (VPGVG)120 at 5 C in a salt-free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3……………………….. 47

1 o 3.5 H NMR COSY spectrum showing assignments of (VPGVG)120 at 5 C in a salt-free aqueous solution. The spectrum was externally referenced to

DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3……………………….. 48

1 o 3.6 H NMR TOCSY spectrum showing assignments of (VPGVG)120 at 5 C in a salt-free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3……………………….. 49

1 13 3.7 H and C NMR HSCQ spectrum showing assignments of (VPGVG)120 at 5 oC in a salt-free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3. f1 on the right axis corresponds to the carbon chemical shit and f2 on the bottom axis corresponds to the proton chemical shift…………………………………… 50

3.8 Chemical shift change as a function of concentration of three salts for (A) the α-protons of glycine, (B) the methyl protons of valine, and (C) the water protons……………………………………………………………………... 51

3.9 Chemical shift change for (A) the amide protons of (VPGVG)120, (B) the amide protons of poly(acrylamide), and (C) the methyl protons of poly(N,N-dimethylacrylamide)……………………………………………. 52

3.10 Residual change after subtraction of the linear portion (c[mM]) in the

chemical shift for the protons from (VPGVG)120 in the presence of NaSCN, plotted together with the residual change in the LCST……………………... 53

3.11 LCST of (VPGVG)120 as a function of pH…………………………………. 55

3.12 Relative chemical shift changes of (VPGVG)120 amide protons as a function of NaSCN. Experiments were performed at (A) 7.7 and (B) at pH 4.0………………………………………………………………………….. 56

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Figure Page

3.13 Relative chemical shift changes of (VPGVG)120 amide proton no. 28

superimposed. Red and green points show changes in relative chemical shift as a function of NaSCN concentration with and without pH 4 control,

respectively………………………………………………………………... 57

3.14 Binding residual from NMR and LCST data for NaI with (VPGVG)120 after the subtraction of the linear salting-out coefficient………………………... 58

4.1 Relative chemical shift changes of N-Ac-GGG-NH2’s α-protons as a

function of salt concentration for five sodium salts………………………… 67

4.2 Relative chemical shift changes of GGG’s α-protons as a function of salt concentration for five sodium salts………………………………………… 69

4.3 Relative chemical shift changes of the aliphatic protons of 50 mM N-Ac- - GGG-CO2 (half-capped GGG) as a function of (A) NaSCN concentration and (B) Na2SO4 concentration. The data points are connected with lines as

a guide to the eye…………………………………………………………... 70

4.4 FTIR spectra of 100 mM N-acetyltriglycinamide in D2O (red) and in 1 M Na2SO4 (black)…………………………………………………………….. 71

4.5 FTIR spectra of 100 mM triglycine in D2O (red) and 1 M Na2SO4 (black)… 72

4.6 Chemical shift changes of the α-protons of 1 mM triglycine as a function

of Na2SO4 concentration. Data points are connected to guide the eye……… 73

4.7 A schematic picture of binding sites of weakly hydrated anions (thiocyanate) and strongly hydrated anions (sulfate) at the capped versus

uncapped triglycine………………………………………………………... 74

5.1 Assortment of molecules. Materials containing α-carbon binding sites of polyglycine or quaternary nitrogens of other compounds could potentially

be designed and incorporated with chromophores or fluorophores like dansyl chloride…………………………………………………………….. 77

5.2 Preliminary results for the effect of NaCl on the disaggregation behavior of

90 mg/mL IgG drug molecules (MEDI528) in the presence of 30 mg/mL PEG3350. Light scattering was measured as function of temperature and

NaCl concentration………………………………………………………… 79

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LIST OF TABLES

Table Page

Abstracted apparent dissociation constants, K ’s, of PDEA from both NMR 2.1 d and LCST data fits. Data were fit using a 95% confidence level. Kd values larger than 1000 mM represent binding weaker than the highest concentration of salt measured…………………………………………….. 26

3.1 Apparent equilibrium dissociation constants abstracted from isotherm fitting of the LCST and NMR data of (VPGVG)120 in the presence of NaSCN…………………………………………………………………….. 54

4.1 Fitted Values for Apparent Dissociation Constants, Kd, for α-Protons in different aqueous salt solutions with GGG and N-Acetyl-GGG-amide. A 95% confidence level was used to extract error bars from the fits. Kd values above 1000 mM represent weaker binding, and blanks were left when no binding could be observed at all within experimental error………………… 68

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ACKNOWLEDGEMENT

I would like to thank my advisor, Professor Paul Cremer, for his support throughout my Ph. D. experience. I learned a lot of science and feel very fortunate to have had the experience to work in his group. I would like to thank my PSU committee members: Scott

Showalter, Will Noid, and Craig Cameron for being productive members on my committee and giving me useful insight on my research. I would also like to thank my former committee from Texas A&M University: Professors Christian Hilty and Hays Rye. Many thanks to my collaborators from the Czech Academy of Sciences in Prague: Professor Pavel Jungwirth, Jana

Hladilkova, and Jan Heyda.

I thank my undergraduate advisors and professors from the University of West Georgia for steering me and preparing me for grad school: John Hansen, Spencer Slattery, Farooq Khan,

Anne Gaquerre, and Swamy Mruthinti.

Last but not least I would like to thank all of my friends, current and former Cremer group members: Dr. Halil Okur, my lab manager Dr. Tinglu Yang, Matt Poyton, Alexis Baxter,

Seung-Yi Lee, Anne Sendecki, Dr. Hudson Pace, Dr. Aaron Robison, Dr. Tao Zhao, Dr. Jaibir

Kherb, Dr. Sarah Flores, Dr. Sean Bowen, Dr. Katherine Cimatu, Sean Bard, Mavreen Tuvilla,

Keturah Odoi, Stephanie Skiles, Yan-Jiun Lee, Dana Coval-Dinant, Dr. Stephanie Preston,

Dave Mertins, Dixie Curley, Tiffani Robsinson, Andrea Benford, Smit Patel, Kandyce Wise,

Lauren Morris, Nathaniel McGinnis, Brittany McGinnis, Melissa Benford, Dewanna Flowers,

Ms. Eunice, Mr Taz, and Melanie McReynolds.

xiii

DEDICATION

I dedicate this dissertation to my parents, Marche’ Rembert and Kelvin Rembert I.

xiv

CHAPTER I

INTRODUCTION

Background and the Hofmeister Series

Proteins are delicately designed biomolecules that serve as the workhorses of biology. They play an essential role in sustaining life with functions extending over a myriad range of physiological processes. A few examples include controlling metabolism and energy production, transporting neurotransmitters and other chemical signals, and regulating blood sugar.1 Associated with the specific task of any particular protein is a unique and complex three-dimensional structure (Figure 1.12). Accordingly, any drastic changes in the structural integrity of the protein will result in loss of its function. Unfortunately, this can be catastrophic.

In fact, many pathologies are directly implicated by protein unfolding or misfolding such as

Parkinson’s, Alzheimer’s, and other neurodegenerative diseases.3–5 It is therefore critical to understand the factors and mechanisms by which proteins become structurally unstable.

On the molecular level, the structures of proteins are dependent on the amide backbone and among the many possible factors that can affect their conformational stability in aqueous solutions, electrolyte effects are quite pronounced. As such, it is of fundamental importance to understand the interactions of ions with protein backbones. In 1888, pharmacology professor

Franz Hofmeister showed that the ability of salts to influence the solubility of aqueous proteins is ion specific and follows a recurring trend which would eventually become known as the

Hofmeister series6,7 (Figure 1.2). Ions to the left decrease protein solubility through a stabilizing effect while ions to the right increase protein solubility through destabilizing effects.

1

Sodium and chloride typically divide the series and both display intermediate behavior.

Curiously, weakly hydrated anions and strongly hydrated cations cause the greatest salting-in effects, while strongly hydrated anions and weakly hydrated cations led to salting-out effects.

Moreover, the effects of anions are typically more pronounced than their cationic counterparts.

Over the last century, the Hofmeister series was shown to extend well beyond protein precipitation studies with occurrence in myriad physico-chemical and bio-chemical phenomena.8 However, even with the widespread occurrence of these effects, no unified molecular picture capable of explaining the series exists to date. In fact, the difficulty in elucidating the underlying mechanisms of the Hofmeister effect is that it occurs at such high salt concentrations, typically in the hundreds of millimolar to molar range.9 Therefore, the key to elucidating the molecular mechanisms of these effects is to use model molecules10 and spectroscopy.

Model Systems

My approach to elucidating the molecular picture of the Hofmeister series on protein backbones has involved employing model systems such as thermoresponsive polymers and oligopeptides (Figure 1.3). The thermoresponsive polymers studied undergo phase transitions at lower critical solution temperatures (LCST) whereby they become insoluble after hydrophobic collapse. Poly(N,N-diethylacrylamide), PDEA, is a biopolymer that serves as a protein mimic by containing both amide groups and hydrophobic moieties. A key feature of this polymer however is that it does not contain an amide NH in its chemical structure. This allows for a direct test for the influence of ion interactions with amides in the absence of NH hydrogen bonding opportunities. Moreover, PDEA is only a hydrogen bond donor and not an

2 acceptor for solvent water molecules which could have pronounced effects on its hydrophobic collapse in the presence of ions. The next thermoresponsive polymer employed was a large

600 residue elastin-like protein, (VPGVG)120, that also undergoes hydrophobic collapse at an

LCST. This net-neutral protein contains no positively or negatively charged amino acid residues and allows for the study of direct interactions of ions with protein backbones and hydrophobic side chains without complication from electrostatic interactions with charged groups.

Small Oligopeptides such as triglycine (GGG) will also serve an important role in understanding the influence of size and the role of electrostatics on ion binding interactions.

Various analogues of the GGG peptide with its N and C termi capped were also studied to systematically follow differences in the behaviors of zwitterionic GGG, neutral end-capped

GGG, and anionic N-capped GGG molecules. They do not undergo LCST phase transitions as does the thermoresponsive polymers discussed above, but the corresponding thermodynamics for these small molecules is still attainable via solubility and partition coefficients as seen in the literature.11–13

Methods

Along with employing the proper model molecules for salt solution studies comes the importance of utilizing the proper thermodynamic and spectroscopic techniques. For thermodynamic phase transition measurements, an automated melting point apparatus with digital image processing will be used (Optimelt). White light scattering intensity of sample capillaries are measured as a function of temperature. The LCST is typically taken as the onset of an increase in light scattering relative to background (Figure 1.4). LCSTs will be measured

3 in the presence of different salts and plotted as a function of salt concentration. Data obtained from the Optimelt device typically provide sufficient signal-to-noise, but in some cases, higher resolution measurements are needed. In this case, dark field microscopy combined with temperature gradient microfluidics provides more precise LCST data.14 Line scans from microscopic images are taken and used to convert pixel positions to temperature. The transition temperature for this type of measurement is taken as the interface between the soluble and insoluble phases of the aqueous polymer solution (Figure 1.5).

Finally, macroscopic LCST data is correlated with spectra acquired from attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. FTIR allows for the investigation of interactions between the amide I (C=O) band of peptide groups and ions. Any direct interactions of ions with the C=O oscillator are expected to show up as changes in the intensity or frequency of this amide I band.

Proton NMR will be used as a complimentary technique to follow ion interactions with every hydrogen atom in the chemical structure of the model molecules employed. For these measurements, we use special NMR tubes that are adapted with coaxial inserts. This allows for the spectral references to be isolated from biomolecule analytes as well as the salts studied.

These types of tubes are especially important for accurately and precisely determining any changes in peak resonances in the biomolecule’s proton spectra (Figure 1.6).

4

Figure 1.1. Ribbon structure of a protein2 illustrating its unique, complex, three-dimensional structure.

5

Figure 1.2. The direct Hofmeister series for cations and anions.

6

Figure 1.3. Model systems employed. Thermoresponsive polymers (left) undergo phase transitions at lower critical solution temperatures (LCST) and serve as suitable protein models for hydrophobic collapse and aggregation. Oligopeptides (right) such as triglycine and other analogues are investigated to determine the influence of charged and non-charged end groups, as well as molecular size, on ion binding.

7

Figure 1.4. A representative light scattering plot as a function of temperature for a thermoresponsive protein or biopolymer measured with an OptiMelt™. The lower critical solution temperature value, LCST, is designated as the onset of the increase in light scattering relative to the baseline.

8

Figure 1.5. A representative dark-field microscope snapshot of various samples in a microfluidic chip on the linear temperature gradient platform. The linescan is taken at the interface between the soluble and insoluble polymer phases (i.e. the transition temperature) and the right figure corresponds to the scattering intensity as a function of pixel position (i.e. temperature).

9

Figure 1.6. Multi-spectra display of a representative proton’s chemical shift in the presence of varying salt concentration.

10

CHAPTER II

AN NH MOIETY IS NOT REQUIRED FOR ANION BINDING TO AMIDES

Introduction

The physical behavior of numerous surfactants, polymers, and biomolecules in aqueous salt solutions is ion-specific and follows a Hofmeister series. 11–13,15–20 The series has been known for over a century and was originally developed as a way of ordering cations and anions by their ability to influence the solubility of egg white proteins.6,7 This ordering, however, was found to recur for a wide variety of proteins along with extending well beyond protein precipitation studies.8 The Hofmeister effect has gotten the attention of many researchers over the last several decades, but unfortunately a unified molecular picture has not yet been developed.16,21–24 The difficulty in elucidating the molecular mechanisms responsible for this series may be a result of the weak interactions between the ions and proteins. In fact,

Hofmeister series behavior is typically found at mM salt concentrations and higher.9 As such, in depth spectroscopic studies are required to elucidate microscopic details, along with complementary macroscopic thermodynamics, to understand the molecular level details of the process and correlate them with macroscopic behavior.

Recent reports have made significant contributions towards developing an understanding of the underlying molecular mechanisms of ion interactions with small organic molecules, model amides, and protein backbones as well as their side chains in aqueous solutions. For instance, it is now known that divalent cations such as Mg2+ and Ca2+ bind very weakly to amide oxygens, while monovalent cations such as Na+ and K+ are excluded.25 If, however, the appropriate negatively charged residues such as aspartic and glutamic acid are present, then

11

Na+ and K+ do form ion pairs with their carboxylate groups.26 Large polarizable ions like I- have been shown to be excluded from the hydration shells of hydrophobic moieties of small organic molecules.27,28 On the other hand, I- as well as SCN- can bind directly to protein backbones.29–31 Specifically, we have shown previously that weakly hydrated anion binding involves a direct interaction with the amide backbone at methylene or other CH groups adjacent to amide moieties. However, NMR measurements and molecular dynamic simulations indicate that the NH group of the amide may participate in anion binding when it was present.

As such, we wished to directly test if it is necessary to have an NH moiety present for the binding of weakly hydrated anions.

PDEA was employed as a model system to probe the necessity of the amide NH site. As can be seen in Figure 2.1, this thermoresponsive polymer is structurally similar to poly(N- isopropylacrylamide) (PNIPAM). The latter is much more widely employed for lower critical solution temperature (LCST) measurements, although both undergo the same inverse phase transition. In fact, PDEA displays an LCST value of ~32°C,32 which is quiet close to that of

PNIPAM (~31 ̊C).33 An important chemical difference for PDEA, however, is that it does not have an NH moiety on its pendant amide group as is the case for PNIPAM or polypeptides.

Therefore PDEA is only a hydrogen bond acceptor in aqueous solutions. To glean molecular level information for ion-specific interactions with PDEA, we have employed NMR and ATR-

FTIR along with LCST measurements as a function of salt identify and concentration. It was found that the ability of a specific anion to modulate the LCST of this polymer in aqueous solution followed a direct Hofmeister series. As such, more strongly hydrated anions salted the polymer out of solution and the more weakly hydrated anions salted it in. Using proton NMR, it was shown that the binding site on PDEA involves the backbone α-protons. Moreover, the

12 apparent equilibrium dissociation constants for weakly hydrated anions were comparable to those for PNIPAM in the presence of the same ions.16,34 ATR FTIR was used to probe the amide I band of PDEA and showed no appreciable changes in peak frequency or intensity in the presence of various salts. This is consistent with the notion that the cations do not interact significantly with the carbonyl moiety of the amide.25 The overall results of this study demonstrate that an NH moiety is not required for the salting in of polymers or peptides by ions in aqueous salt solutions.

Materials and Methods

Lower Critical Solution Temperature Measurements.

Lower critical solution temperature measurements of PDEA were performed by measuring the turbidity change of polymer solutions in capillary tubes by a CCD camera that was interfaced with digital image processing software (MPA 100 Optimelt automated melting point system, Stanford Research Systems). The light scattering intensity was measured as a function of temperature at a ramp rate of 1.0 °C per minute. The transition temperatures were determined as the onset of the increase in light scattering intensity relative to baseline as described previously.19,35

Sample Preparation.

Poly(N,N-diethylacrylamide) with a molecular weight of 260 kDa was purchased from

Polymer Source Inc. (Dorval(Montreal), Quebec, Canada). Sodium salts were purchased from

Sigma Aldrich (St. Louis, MO) and used as received. NaSCN was at least 98% pure while the other salts used, Na2SO4, Na2CO3, NaS2O3, NaH2PO4, NaF, NaCl, NaBr, NaNO3, NaClO4, and

13

NaI, were at least 99% pure. 18.2 MΩ·cm purified water from a Thermo Scientific Nano pure

Barnstead purification system was used to prepare salt and polymer solutions. For LCST and

NMR measurements, the polymer was precipitated from hot aqueous solution to remove unpolymerized monomers and short chain oligomers. Redissolved aqueous polymer solutions were aliquoted and vacuum dried from a 10 mg/mL stock solution. The appropriate concentrations of salt solutions were added to the vacuum dried pellets at temperatures below the lower critical solution temperature, followed by vortexing until the polymer was fully dissolved. The final PDEA concentration employed in both LCST and NMR measurements was 1 mg/mL.

NMR Measurements.

All spectra were acquired on a 400 MHz NMR spectrometer equipped with a 5 mm TXI probe (Bruker, Billerica, MA) at a temperature of 5°C. For chemical shift assignments of

PDEA, spectra employed were [1H,1H]-NOSEY (100 ms mixing time) and [1H,1H]-TOCSY

(100 ms mixing time).36 TopSpin software (Bruker) was used for data processing. For titrations of PDEA with salts, 1H NMR spectra were acquired using Watergate for water suppression.37

Sample spectra were externally referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate

(DSS) (Cambridge Isotope Laboratories) in pure D2O (99.9% D, Cambridge Isotope

Laboratories) in NMR tubes adapted with coaxial inserts (Wilmad-LabGlass). The DSS was always in the inner of the concentric tubes, while the polymer sample was in the outer tube.

The DSS reference was never exposed to the polymer or varying salt concentrations.

14

FTIR Measurements.

Infrared spectra were measured with a Nicolet 470 FTIR spectrometer using an attenuated total refection attachment (Pike Miracle, Madison, WI).25,38 The ATR crystal (diamond coated

ZnSe, Pike Technologies, Madison, WI), was employed in a single bounce geometry to make the measurements. The infrared radiation was detected with a liquid nitrogen cooled MCT detector (Thermo Electron Corp., Madison, WI). All spectra were averaged over 128 scans and collected in a range from 650 cm-1 to 4000 cm-1 with a spectral resolution of 2 cm-1. The samples for ATR-FTIR were prepared by dissolving vacuum dried PDEA in the desired salt solution. The final polymer concentration was 10 mg/ml. To completely dissolve the polymer, the samples were stored overnight at 4 oC. During acquisition of spectra, the sample stage was kept at 5 °C which was at least 10 °C below the LCST of the polymer under all conditions.

Sample spectra were measured a minimum of three times each. Moreover, an otherwise identical salt solution without PDEA was used as to make background measurements, which were made immediately preceding sample measurements. This background was subtracted from each sample spectrum using OMNIC software.

Results

Solubility of PDEA as a Function of Sodium Salts.

Figure 2.2 A plots the LCST of PDEA as a function of salt concentration for eleven sodium salts. The transition temperature decreases linearly with increasing salt concentration for solutions containing more strongly hydrated anions. The ability of a particular sodium salt to

2- 2- 2- - - - decrease the LCST followed the rank ordering: CO3 ~ SO4 > S2O3 > H2PO4 > F > Cl . By

- - - - - contrast, when Br , NO3 , ClO4 , I , and SCN were added to solution, the transition temperature

15 changed in a nonlinear fashion. These nonlinear data can be fitted to a simple empirical equation consisting of a linear term plus a Langmuir isotherm term (eqn. 2.1):16,34

Tmax[M ] LCST(°C)  T0  c[M ] eqn. 2.1 K D [M ]

o T0, 31.9 C, is the LCST in the absence of salt, c, which has units of temperature/concentration

(oC/[M]), is a linear term that represents a salting-out coefficient and can be attributed to increases in the surface tension at the hydrophobic moieties/water interface as well as excluded volume effects from the depletion of ions from the polymer/water interface.39 The non-linear term represents an apparent binding isotherm with the coefficient Tmax as the maximum increase in the LCST of the polymer upon saturation binding of ions. Moreover, Kd is the apparent equilibrium dissociation constant of the anion-polymer interaction. The data for more strongly hydrated anions can also be fit to equation 1 assuming an infinitely weak binding interaction; e.g. the non- linear term would approach zero as Kd approaches infinity. After subtracting the linear term (T0 + c[M]) from the data in Figure 2.2 A for weakly hydrated anions, the residual changes in LCST could be plotted as a function of salt concentration

(Figure 2.2 B). The residual LCSTs show saturation behavior and can be fit as Langmuir isotherms as shown by the solid line fits to the data. The corresponding apparent Kd values are listed in Table 2.1.

NMR titration of PDEA versus sodium salts.

In a set of proton NMR experiments, Na2SO4, NaCl, NaI, and NaSCN were chosen as representative Hofmeister salts to probe interactions of PDEA with anions. Figure 2.3 plots the

16 relative chemical shift changes (Δδ) of the N-CH2 protons (black circles), methyl protons (green circles), and backbone α-protons (red circles) as a function of salt concentration. As can be seen from the data in Figures 2.3 (A) and (B), the relative chemical shift decreases linearly for all protons as a function of increasing concentrations of Na2SO4 and NaCl. The linear decreases in chemical shift resemble the linear decreases in LCST for the corresponding salts in Figure

2.2 (A). As such, having all linearly decreasing proton chemical shifts in the presence of

29 Na2SO4 and NaCl is consistent with the thermodynamic salting-out of the polymer. Figures

2.3 (C) and (D) plot the relative chemical shift changes of N-CH2, methyl, and α-protons with increasing concentrations of NaI and NaSCN, respectively. The three types of protons display distinct behavior. Specifically, the chemical shift of N-CH2 and methyl protons linearly decreases with increasing concentrations of salt, while the α-protons shift nonlinearly. Such nonlinearity of the chemical shift is consist with a binding interaction between ions and the polymer at the site of the -protons and the corresponding -carbon. In fact, this result is also consistent with our previously reported binding site of SCN- and I- to the backbone α-protons

29 of an elastin-like polypeptide. Moreover, linear decreases in the chemical shift of the N-CH2 and methyl protons suggest that these sites do not significantly contribute to the salting-in of the polymer. The chemical shift changes of PDEA’s α-protons as a function of NaSCN and

NaI can be fit to eqn. 2.2:

 [M ]  ( ppm)  c[M ] max eqn. 2.2 KD [M ]

Eqn. 2.2 is analogous to eqn. 2.1 and has a linear and nonlinear term where now the coefficient c has units of chemical shift per molarity of salt added. The non-linear term again

17 represents direct anion binding with the coefficient, δmax, representing the maximum increase of the relative chemical shift change upon saturation ion binding, and Kd is again the apparent equilibrium dissociation constant. After subtraction of the linear portion of the data for the - protons, the residual changes in the chemical shift follows a binding curve for both NaSCN and NaI (Figure 2.4). The residual non-linear portions of the LCST data from Figure 2.2 (B) for NaSCN and NaI are also shown. As can be seen NaSCN binds slightly tighter to the polymer than NaI for both NMR and LCST data. The abstracted apparent equilibrium Kd values from both fitted data sets are shown in Table 2.1. Overall, the binding constants from

LCST measurements were weaker than those of the corresponding proton NMR measurements. It should be noted however that while the Kd’s from LCST data are averages of interactions of ions over the entire polymer surface, the Kd’s reported from NMR data represent a single site of interaction i.e. the α-proton. Moreover, the spectroscopic binding constants are within the same order of magnitude as the thermodynamic binding constants. As such, this shows that the salting-in behavior of PDEA in presence of both NaSCN and NaI is a result of ions binding to the backbone α-protons.

FTIR of PDEA versus Sodium Salts.

In a final set of experiments, FTIR measurements were made to probe the interactions of various sodium salts with the carbonyl group of PDEA. Figure 2.5 shows the spectrum of the amide I region of PDEA in D2O. It should be noted that the amide I band of PDEA arises

40 mostly from the carbonyl stretch in D2O, yet the peak shape is somewhat different compared with PNIPAM.41 Specifically, PDEA gave rise to two absorption peaks centered at 1619 and

1599 cm-1 below its LCST,42 while PNIPAM only has one amide band. Figure 2.6 shows the

18 carbonyl peak position of both the 1619 cm-1 peak and the 1599 cm-1 peak of PDEA as a function of salt concentrations for 4 different anions. As can be seen, both peak positions remained essentially the same within the experimental error. Such FTIR data suggests that there is little, if any, direct interactions between the sodium salts and the carbonyl groups. Such results are in agreement with previous NMR, FTIR, and MD simulation data for amides, where such interactions were found to be unfavorable.25,30,31,43,44

Discussion & Conclusion

As illustrated above, the absence of an amide NH moiety has no significant effect on the binding of Hofmeister anions to amide-based polymers or their corresponding salting-in

- - - - behavior. This is clearly demonstrated by the salting in of PDEA by SCN , ClO4 , I , NO3 , and

Br - ions. If these weakly hydrated ions did not interact directly with PDEA, the LCST of the polymer would only be expected to display a linear decrease. Instead, favorable anion interactions should increase the solvent accessible surface area of the polymer.39 This leads to salting-in and nonlinear changes in the LCST in the presence of more weakly hydrated anions.

Further increases in the concentration of even weakly hydrated ions eventually leads to salting- out and linear decreases in the LCST. The reported dissociation constants and salting out coefficients for PDEA are quite close in value to those for other poly(N-alkylacrylamide) based polymers including PNIPAM,16,34 and poly(vinylpyrrolidinone)45 which suggests that the amide NH interacts, at best, very weakly with anions in aqueous solutions. Indeed, these three systems all have similar α-proton hybrid binding sites in common which suggests that the structural location of the amide, whether in a protein backbone or on a polymer side chain, does not have any significant effects on the ability of anions to bind. There might however be

19 differences in the mechanisms of hydrophobic collapse. It was shown that the salting-out of

PNIPAM and ELPs correlated directly with surface tension for weakly hydrated anions and ion hydration entropy for strongly hydrated anions. While the salting-out behavior of PDEA showed the same correlation between weakly hydrated anions and surface tension (Figure 2.8), the strongly hydrated anions showed a correlation with not only ion hydration entropy, but also roughly to air water interface surface tension increments (Figures 2.7 and 2.8). This suggests that significant contributions from both mechanisms are involved with the hydrophobic collapse and salting-out of the polymer by strongly hydrated anions. Moreover, these effects may be, in part, due to the fact that PDEA is only a hydrogen bond acceptor to the solvent water molecules and also cannot form neither intra nor intermolecular hydrogen bonds with itself upon collapse.

In terms of the hybrid amide dipole/ aliphatic-carbon sites, N-CH2, methyl, and backbone

β-protons were also expected to display binding behavior. Curiously, there was no apparent binding observed at either of these moieties. N-CH2 groups may be sterically inaccessible while it is possible that both methyl and backbone β-protons were not electro-positive enough from the inductive effects of the electron withdrawing amide. In the case of ELPs or other peptides, there are two amide dipoles withdrawing electron density from the adjacent aliphatic carbons.

This is not the case for PDEA. β-protons may also have been affected by the tacticity of the chiral backbone α-carbon binding site. Stereochemistry at these types of sites determines the accessibility to solvent as well as the ability of ions to bind.46

Overall, it is not surprising that the NH site is not of great significance. Indeed, an amide

NH should be hydrogen bonded to water in aqueous solution. In order for an anion to interact with this site, both this amide-water as well as the anion’s hydration shell need to be disrupted.

20

On the other hand, interactions between the -carbon and the anion are more favorable. For this type of interaction, no specific hydrogen bonds around the polymer need to be disrupted, just its hydrophobic hydration waters as well as the hydration shell of the anion. This is evidently the key interaction as demonstrated by these experiments. In this case the -carbon should bear a slight positive change due to an induction effect from the adjacent electron withdrawing amide moiety.29–31 As such, it should attract the anion on electrostatic grounds.

Such an interaction is quite weak, but its formation pays only a small penalty in terms of specific hydrogen bond breaking compared with interactions with the amide NH.

21

Figure 2.1. Monomeric structures of Poly(N-isopropylacrylamide), PNIPAM, and Poly(N,N- diethylacrylamide), PDEA.

22

10 (A) 36 (B) NaSCN NaSCN NaI NaI 8 32 NaClO NaClO 4 ) 4

NaNO C 3 NaNO

o 3

) 28 NaBr ( 6 NaBr

C

O NaCl NaCl ( NaF 24 NaH PO 2 4 4 Na S O 2 2 3 LCST 20 Na SO 2 4 2 Na CO 2 3

16 LCST - c*[M] 0 12 0 200 400 600 800 1000 0 200 400 600 800 1000 Concentration (mM) Concentration (mM)

Figure 2.2. (A) LCSTs of PDEA as function of salt concentration for eleven sodium salts. Data were best fit linear or to an expression containing both a linear and nonlinear binding term (see eqn. 1). (B) Residual LCSTs of PDEA versus 6 salts after subtraction of the linear component. The data for NaSCN, NaI, NaClO4, NaNO3, and NaBr were best fit to a binding isotherm. The data points for NaCl are connected with lines as a guide to the eye.

23

Figure 2.3. Relative chemical shift changes (∆δ) of N-CH2, Alpha, and Methyl protons of PDEA as a function of salt concentration for Na2SO4 (upper left), NaCl (upper right), NaI (lower left), and NaSCN (lower right). Data were best fit linear or to an expression containing both a linear and nonlinear binding term (see eqn. 2). Structure of PDEA monomer with circles adjacent indicating their corresponding protons on graphs.

24

0.025 12

LCST - T 10 0.020

8 0.015

0

- c*[M] 6 0.010 4

- c*[M] (ppm)

Residual -proton vs. NaSCN (

o

 0.005 C Residual -proton vs. NaI 2 Residual LCST vs. NaSCN ) Residual LCST vs. NaI 0.000 0 0 200 400 600 800 1000 Concentration (mM)

Figure 2.4. Residual change after subtraction of the linear portion in the chemical shift changes for backbone α-protons, left y-axis (black), and residual LCST changes, right y-axis (blue), both as a function of either NaI or NaSCN concentration.

25

Table 2.1. Abstracted apparent dissociation constants, Kd’s, of PDEA from both NMR and LCST data fits. Kd values larger than 1000 mM represent binding weaker than the highest concentration of salt measured.

26

Figure 2.5. FTIR spectrum of PDEA in D2O at 5 °C, with individual Gaussian peaks fits (green plots) and cumulative peak fit (red plot).

27

(A) (B)

Figure 2.6. The individual amide I peak positions plotted as a function of employed salt concentrations for (A) 1599 cm-1 and (B) 1619 cm-1 peak positions. The yellow highlighted -1 region represents the 2 cm resolution of the spectrometer centered at the amide I band in D2O in the absence of salt.

28

0 - - SCN Br - I- NO -10 3 - Cl- ClO 4 -20 ) F-

- -30 H2PO4

C/mol

o -40

(

c 2- S2O3 -50 CO 2- 3 -60 SO 2- 4 0 50 100 150 200 250

- Shydr (J/K mol)

Figure 2.7. Correlation between the salting-out coefficients, c, from data fits to eqn.1 as a function of the hydration entropy of the eleven Hofmeister anions16 used in this study. The red line is a linear fit for values corresponding to the strongly hydrated anions. The salting out coefficients for strongly hydrated anions show a general dependence on the hydration thermodynamics of the anions while the weakly hydrated anions do not.

29

0 SCN- I- Br- Cl- -10 - NO - 3 ClO 4 -20

) F- - -30 H2PO4

C/mol -40

o ( 2- S2O3 c -50 2- CO3 -60 2- SO4

-70 0.5 1.0 1.5 2.0 2.5 3.0  (mN L/m mol)

Figure 2.8. Correlation between the salting-out coefficients, c, from data fits to eqn.1 as a function of the surface tension increments for the eleven Hofmeister anions16 used in this study. The green and red lines are both linear fits to the strongly hydrated and weakly hydrated anion values, respectively. The salting out coefficients for both sets of anions show a general dependence on the air/water interface surface tension increments.

30

CHAPTER III

MOLECULAR MECHANISMS OF ANION-SPECIFIC BINDING TO PROTEIN

BACKBONES

Introduction

The influence of electrolytes on biological function is ubiquitous, with specific choices of salt ions for particular tasks in living organisms being crucial.47–49 For example, the cytosol is rich in potassium but poor in sodium, while the opposite is true for extracellular liquids.50

Sodium exhibits slightly stronger affinity for proteins than potassium and could thus interfere at higher concentrations with the function of cellular enzymes.51 Chloride, which interacts very weakly with proteins, is the universally present anion in biology.50 By contrast, iodide, which is much rarer, as well as the toxic anions, perchlorate and thiocyanate, interact strongly with proteins, particularly in the thyroid. The latter two anions are able to replace iodide and thus compromise thyroidal function.52,53 Understanding the tight regulation and toxicity of various ions has been difficult due to the lack of identification of the molecular-level binding sites.

Previous studies have shown that the behavior of proteins and other polymers in aqueous salt solutions often follows a regular pattern that was first identified over 120 years ago.7,10,47,49,54

This recurring trend in ion specificity is known as the Hofmeister series. Over the past half century, numerous efforts have been made to correlate the differences in the solubility of proteins in salt solutions to the various ion sizes, geometries, charge-to-volume ratios, entropies of hydration, and related physical properties.10,11,55–58 The effects of the ions in the

Hofmeister series are, however, typically manifested only above hundereds of millimolar salt

31 concentration. This is relatively weak compared with numerous other biological interactions, which has made it hard to pinpoint the specific binding sites for ion−protein interactions. To overcome this difficulty, we have undertaken a series of thermodynamic and proton NMR experiments on a model system consisting of the pentameric amino acid repeat, VPGVG. This biopolymer, known as an elastin-like polypeptide (ELP), is subject to an inverse phase transition as a function of temperature.59 At low temperature it is soluble in water, while above its lower critical solution temperature (LCST) the molecule undergoes hydrophobic collapse and becomes insoluble. The particular ELP employed in these experiments was (VPGVG)120.

Moreover, the macromolecule consisted of 120 pentameric repeats for a total of 600 residues, in addition to a very short leader (SKGPG) and trailer (WP) sequence. This ELP was investigated in a set of salt solution studies and displayed an LCST of ~31 °C in pure water.

This phase transition temperature is known to be decreased by sodium salts in the following order:17

2- 2- 2------CO3 > SO4 > S2O3 > H2PO4 > F > Cl > Br > NO3 > I > ClO4 > SCN

Anions to the left of chloride are strongly hydrated and lead to the hydrophobic collapse of the

ELP, causing it to salt-out of solution. On the other hand, anions to the right of chloride are weakly hydrated and help keep the biopolymer in solution in its uncollapsed state. Chloride typically divides the anionic series and displays intermediate behavior.

In the current study, we showed that increases in ELP solubility in the presence of large soft anions correspond to nonlinear changes in the relative proton chemical shifts at the α- proton and β-proton positions of the polypeptide as a result of anion binding. Decreases in ELP

32 solubility when more strongly hydrated anions were introduced to solution corresponded to linear proton chemical shifts. The hydrophobic side chains of valine do not exhibit any appreciable affinity to any of the investigated ions (Figure 3.1). These experimental results were complemented and confirmed by molecular dynamics (MD) simulations of a VPGVG pentapeptide; the MD results are not presented in this dissertation but can be found in the literature.29 The present study also revealed a novel hybrid binding site composed of polar and nonpolar groups at the peptide backbone. Such findings should aid general predictions of the molecular-level behavior of proteins in aqueous salt solutions.

Materials and Methods.

ELP Overexpression and Purification

The details of ELP overexpression and purification are reported elsewhere.60,61 Briefly,

(VPGVG)120 was overexpressed in BLR/DE3 Escherichia coli in TB medium (Mo Bio TB dry high nutrient growth media). Purification of (VPGVG)120 was accomplished by sonication of the cells to lyse them, followed by two rounds of inverse transition cycling, whereby the ELP precipitated above its phase transition temperature. Samples were then dialyzed for 2 days against purified water with a minimum resistivity of 18.2 MΩ·cm using a Thermo Scientific

Barnstead Nano pure water purification system. The concentration of (VPGVG)120 was determined by measuring the absorbance at 280 nm, where ε = 5690 M−1 cm−1.

Lower Critical Solution Temperature Measurements

The LCST was measured by turbidity on an automated melting point device with digital image processing software (MPA 100 Optimelt™ automated melting point system, Stanford

33

Research Systems). The intensity of light scattering was measured as a function of temperature using a ramp rate of 0.5 °C/min. The LCST values given herein corresponded to the onset of the increase in light scattering relative to the baseline.19,35

NMR Measurements.

NMR spectra for titrations of ELP with sodium salts were obtained on a 400 MHz NMR spectrometer equipped with a 5 mm TXI probe. 1H spectra were acquired using W5

WATERGATE37 or presaturation for water suppression. All NMR samples were measured at

5 oC, which is below the phase transition temperature of the ELP in all cases that were studied.

Sample spectra were externally referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate

(DSS) (Cambridge Isotope Laboratories) in pure D2O (99.9% D, Cambridge Isotope

Laboratories) in NMR tubes adapted with coaxial inserts (Wilmad-LabGlass). The DSS was always in the inner of the concentric tubes, while the ELP sample was in the outer tube. As such, the DSS control was never exposed to the ELP or varying salt concentrations. Spectra for the chemical shift assignments of the biopolymer were acquired on the 400 MHz spectrometer and on a 500 MHz spectrometer with a TCI CryoProbe (Bruker, Billerica, MA).

Spectral assignements were obtained in [1H,1H]-NOSEY (100 ms mixing time) (Figure 3.4) , double quantum filtered [1H,1H]-COSY (Figure 3.5), and [1H,1H]-TOCSY (100 ms mixing time) experiments (Figure 3.6),36 as well as with [1H,13C]-HSQC (Figure 3.7) and multiple

[1H,13C]-HMBC (J-filter ranges between 4 and 20 Hz).62 TopSpin software (Bruker) and

Mnova software (Santiago de Compostela, Spain) were used for data processing.

34

Sample Preparation.

The NaSCN, NaI, and Na2SO4 employed in these experiments were purchased from Sigma

Aldrich (St. Louis, MO), while NaCl came from VWR (Radnor, PA). All salts were at least

98% pure, and employing even higher purity salts did not change the NMR spectra or the LCST data. Poly(acrylamide) was purchased from Aldrich. Poly(N,N-dimethylacrylamide) was synthesized by the group of David E. Bergbreiter at Texas A&M. Salt and polymer solutions were prepared with 18 MΩ·cm purified water. Stock solutions of salts and ELPs were prepared at double the desired concentration and mixed 1:1 (volume to volume) to obtain the desired sample concentration. The total ELP V5-120 polymer concentration in all experiments was 3.5 mg/mL (70 μM). This corresponds to an amino acid monomer concentration of ∼42 mM.

Control experiments with poly(N,N-dimethylacrylamide) and poly(acrylamide) were also performed at concentrations such that the corresponding monomer concentrations were ∼42 mM.

Results.

(VPGVG)120 Solubility as a Function of Salt Concentration

Figure 3.2 plots the change in the LCST (ΔLCST) of the ELP as a function of salt concentration for NaSCN, NaCl, and Na2SO4. As can be seen, the LCST decreases linearly with increasing concentrations of NaCl and Na2SO4. By contrast, NaSCN leads to a nonlinear increase in the LCST. The data for NaSCN can be fit by eqn. 1, which consists of a linear term plus a binding isotherm:16,34

T [M ] LCST (°C)  c[M ]  max eqn. 3.1 Kd [M ]

35

In the first term, the coefficient, c, represents salting-out and has units of temperature divided by molar concentration (oC/[M]). The second term, the binding isotherm, contains the apparent equilibrium dissociation constant, Kd, as well as the constant Tmax, which represents the maximum increase in the LCST caused by the binding of ions. As such, Tmax has units of

o temperature ( C), while Kd is in units of molar concentration ([M]). The data for NaCl and

Na2SO4 can also be fit to eq. 1 by assuming an infinitely weak binding interaction (i.e. Kd would approach infinity). In thermodynamic models, salting-in has been associated with ion accumulation at the macromolecule/water interface, while salting-out has been associated with ion depletion and excluded volume effects.39

(VPGVG)120 Binding Sites Probed by NMR

To probe the molecular-level mechanism for changes in the phase transition temperature with salts, proton NMR experiments were conducted with the same three salts at a constant temperature of 5 °C, which is below the LCST of the ELP under all conditions. Figure 3.3 shows the one dimensional assignments of the protons in (VPGVG)120 in pure water. As salt was added to solution, different protons in the spectrum displayed markedly different behavior.

The changes in the chemical shift (Δ δ) for the α-protons of the glycine residues are plotted in

Figure 3.8 (A). As can be seen, these protons shifted in a fashion that was reminiscent of the thermodynamic LCST data. Specifically, in the presence of NaSCN, the change in the chemical shift displayed markedly nonlinear behavior. Such a result is consistent with the notion that

SCN− and, to a much lesser extent, Cl− interact directly with the methylene units in the polypeptide backbone. The data for the chemical shift were fit to eqn. 3.2:

36

 [M ]  ( ppm)  c[M ] max eqn. 3.2 Kd [M ]

The form of this equation is identical to that of eqn 3.1, where the left-hand side is now the change in chemical shift, Δδ, with respect to salt-free sample. In this case, the coefficient, c, has units of chemical shift divided by molar concentration (δ/[M]), whereas Δδmax is the maximum change in the chemical shift for the nonlinear portion of the curve at saturation. By contrast to the data in Figure 3.8 (A), the methyl protons in Figure 3.8 (B) from the valine residues displayed only linear changes in their chemical shift for all three sodium salts. The proton chemical shift for water protons63 also varied in a linear fashion. The linear shift in the water protons as well as those on hydrophobic side chains is consistent with the linear salting- out effect from the thermodynamic data in Figure 3.2. Moreover, the linear salting-out of the

ELP can be correlated with increasing surface tension at the polymer/water interface when the sodium salts of weakly hydrated anions like SCN- and Cl- are added to solution.16 For strongly

2- hydrated SO4 anions, the salting-out behavior has been shown to correlate with the entropy of hydration of the anions. This is essentially an excluded volume effect, since well-hydrated anions remain in the bulk solution and are therefore depleted from the polypeptide/water interface. An additional potential site for interactions of weak hydrated anions involves the NH protons of the amide groups. The addition of NaSCN leads to a decidedly nonlinear change in the chemical shift of the backbone NH protons shown in Figure 3. 9 (A). This is expected as the NH protons and α-protons are in spatial proximity. By contrast, NaCl led to much less curvature in the chemical shift change, while virtually none was observed for Na2SO4. Since the data in Figure 3.9 (A) appear to indicate that there are interactions between SCN− and the amide NH protons, it is important to probe these putative interactions in the absence of an

37 adjacent aliphatic group that can act as a binding site. To do this, control experiments were performed with poly(acrylamide) in water using NaSCN. As can be seen in Figure 3.9 (B), the nonlinearity of the chemical shift becomes much less pronounced. Upon methylation of poly(acrylamide) to poly(N,N-dimethylacrylamide), however, the chemical shift of the methyl protons shift in nonlinearly and again show pronounced binding behavior in Figure 3.9 (C).

Such a result is consistent with previous thermodynamic studies, which indicated that the apparent binding constants for weakly hydrated anions are at least 2 orders of magnitude tighter when at least one of the NH groups of the acrylamide side chain is methylated.16,64 Although the data described above indicate that the binding site for weakly hydrated anions involves the

α-position of the polypeptide, there are probably varying degrees of interactions with multiple sites. Chemical shift data were collected for all aliphatic protons on the ELP. In addition to deviations from linearity for protons associated with the α-carbons, less pronounced curvature also could be seen from β-protons and the proline ring. These effects can be displayed by subtracting the linear portion of the chemical shift changes and plotting the residuals (Figure

3.10). All data fit well to a simple binding isotherm. The apparent equilibrium dissociation constants for the varying sites ranged from ∼50 mM for the α-protons of glycine and the methylene adjacent to amide on the proline ring to ∼270mM for the β-protons and other proline ring methylene groups. The dissociation constant approached infinity for the γ-protons from the methyl groups of the valine residues. The apparent equilibrium dissociation constants from isotherm fitting are provided in Table 3.1. One can also abstract the nonlinear portion of the

LCST data for the ELP in the presence of varying concentrations of NaSCN. This residual data is also plotted in Figure 3.10 (blue squares along with an arrow to the right), and the y-axis in this case involves ΔLCST instead of Δδ. The apparent Kd value from the LCST data is 390

38 mM, which should correspond to an averaged value of interactions over the entire polymer surface. It is slightly weaker than the values abstracted from NMR because thermodynamic measurements were made at the LCST of the ELP, which was always higher in temperature. It should be noted that the identical NMR and LCST measurements used for these three salts were repeated with NaI.29 The results were nearly identical with those of NaSCN, although the

I− interacted more weakly with the amide NH proton.

Additional control measurements with pH.

The pH of the (VPGVG)120 polymer solutions with no added salt and with 1M NaSCN, 1M

NaCl, and 0.25M Na2SO4 was measured to be 7.8, 7.7, 7.6, and 7.7 respectively. As such, the pH was essentially unchanged under all experimental conditions shown in Figures 3.2, 3.8, and

3.9. It should be noted that the valine, proline, and glycine have no titratable groups. The N- and C- termini of the 600 residue ELP however were charged and there was also a very short lead (SKGPG) and trailer sequence (WP) at each end, of which it contains one lysine.60,61 As such, LCST experiments were performed as a function of pH as shown in Figure 3.11. The phase transition temperature was insensitive to pH over a range from pH 4 to 10 (We thank

Dr. Jaibir Kherb at the University of Delhi, India for assistance with these measurements).

It should be noted that in NMR experiments, the NH protons can undergo exchange with the bulk solution. Under some conditions, this can be on nearly the same time scale as the

NMR experiment. By contrast, the exchange rate is greatly slowed near pH 4 compared with the NMR timescale.36 Therefore, the experiments shown in Figure 3.9 (A) were repeated at pH

4.0 as a control and can be seen in Figure 3.12. These low pH measurements were made in 10 mM sodium citrate buffer. As can be seen, the NMR shifts of the four amide protons are quite

39 similar for near neutral and acidic pH (data are also shown at pH 7.7 for a side-by-side comparison with 4.0). Moreover, Figure 3.13 places the data from amide proton resonance no.

28 at pH 7.7 and 4.0 directly on top of each other, where the overlap is seen to be very good.

It is this resonance that was used to plot the data in Figure 3.9 (A) and, therefore, potential problems from proton exchange should be minimal.

Discussion

The NMR and MD simulation data29 clearly demonstrate that the salting-in effect of

NaSCN is primarily caused by the binding of weakly hydrated SCN- anions with the

− polypeptide backbone of the ELP. The preferred interaction of SCN with CHn groups next to atoms like nitrogen and the carbonyl carbon is connected with the electron-withdrawing ability and inductive effects of these atoms from the neighboring groups. Due to this effect, the overall charge on the CHn groups corresponding to α-carbon atoms and the proline heteroring is slightly positive (around +0.1 e), while that of the CH3 groups on hydrophobic valine side chains is weakly negative (−0.08 e), as calculated by the force field used in the simulations of our collaborators.29,65 The partial formal positive charge on the amide nitrogen and carbon naturally enhances the binding of large, soft anions, such as SCN−. A similar effect is also observed for iodide as shown in Figure 3.14. In addition, hydrogen bonding to the neighboring

− - amide NH proton groups helps the interaction of SCN and I with the adjacent CHn group, but it is not critical. This was demonstrated by our collaborators simulation where all the backbone

NH protons were replaced with methyl groups.29 Such methylation prevents the formation of amide proton hydrogen bonds with SCN−. Nonetheless, the overall amount of SCN− binding to the protein is only weakly affected. Therefore, the binding site for backbone amide groups

40 is an inseparable combination of polar and nonpolar moieties. These hybrid hydrophobic/hydrophilic binding site does not require specific hydrogen bonds between water and the polypeptide to be broken, since water only hydrophobically hydrates the methylene units without strong OH directionality in the absence of salt. In contrast, binding between anions and the amide NH proton requires breaking of specific water−peptide hydrogen bonds and would therefore be thermodynamically less favorable on enthalpic grounds. The idea that weakly hydrated anions do not bind appreciably to generic alkyl chains is in good agreement with previous thermodynamic studies that showed that hydrophobic moieties lead to salting- out of molecules, even in the presence of the most weakly hydrated anions.17 Indeed, weakly hydrated anions do not bind appreciably with methyl groups that are neither directly bonded to an electron withdrawing atom nor to the next nearest neighbors with an electron withdrawing atom. Such a result is somewhat analogous to the findings for weakly hydrated anions at the air/aqueous interface, although the polymer/aqueous interface is considerably more complex.66,67 The amide backbone binding sites for weakly hydrated anions are the most significant locations for the salting-in of uncharged polypeptides. Such binding will lead to a saturation binding isotherm. However, even the most weakly hydrated anions also can cause salting-out behavior by increasing the surface tension of the purely hydrophobic portions of the polymer/water interface.10,16,17,34 In other words, specific binding sites lead to net anion accumulation, but upon saturating all those sites, further increases in salt concentration actually lead to net anion exclusion. Therefore, weakly hydrated anions display both salting-in and salting-out behavior. By contrast, the interactions of strongly hydrated anions with uncharged and nonpolar residues on the peptide chain only lead to salting-out effects. Their exclusion from the polypeptide/water interface leads to hydrophobic collapse and aggregation on

41 entropic grounds through an excluded volume effect.39 This is similar to the behavior of most cations, such as Na+, which are also strongly excluded from the polypeptide backbone.25,43 The general exclusion of most cations from the polymer/water interface is the reason that specific cation effects are far less pronounced than anion effects. However, both strongly hydrated anions and cations can, however, bind appreciably with charged protein side chains,30,31 and this could lead to salting-in behavior if the appropriate residues are present.18,26

Conclusions

The solubility and aggregation behavior of organic molecules in aqueous solutions has been traditionally assumed to depend upon the separate contributions of charged, polar, and hydrophobic groups. However, we have demonstrated that anion binding to polypeptide backbones is dominated by the unique nature of the nonpolar/polar α-carbon sites. Such interactions, along with ion pairing 49,51,68in the presence of charged residues, dominate the

Hofmeister chemistry of large proteins. Beyond Hofmeister effects, the phase behavior of macromolecules and amphiphiles can be tuned by exploiting the unique properties of hybrid polar/nonpolar sites. Nature almost certainly does this. For example, one of the strongest protein stabilizers in vivo is trimethylamine N-oxide, which contains three modestly hydrophilic methyl groups directly bound to the nitrogen atom of an NO moiety.69 These methyl groups actually orient away from hydrophobic interfaces,70 which may help explain this osmolyte’s ability to stabilize the structure of folded proteins. A synthetic example of a hybrid polar/nonpolar site comes from the cyclic cavities of triazophanes71 and concavities,72 which can capture anions. Knowledge of how to synthesize and exploit hybrid polar/nonpolar

42 sites may allow the creation of new materials, sensor devices, and novel self-assembled structures.

43

Figure 3.1. Schematic diagram depicting the molecular-level binding sites for SCN− with (VPGVG)120. As shown, the anion binds preferentially to the Glycine methylene moiety within the amide backbone, but not appreciably to a purely hydrophobic methyl group such as the ones on valine. The anion also binds to some extent with the NH proton. It should be noted that only one resonance structure of SCN− is depicted. The other one consists of two double bonds and the placement of the formal charge on the nitrogen atom.

44

Figure 3.2. Change in LCST (∆LCST) of (VPGVG)120 as a function of salt concentration for o Na2SO4, NaCl, and NaSCN. ∆LCST=0, 31.9 C, corresponds to the LCST of the polymer in the absence of salt.

45

o Figure 3.3. Proton NMR spectrum showing assignments of (VPGVG)120 at 5 C in a salt-free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide above.

46

1 o Figure 3.4. H NMR NOESY spectrum showing assignments of (VPGVG)120 at 5 C in a salt- free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3.

47

1 o Figure 3.5. H NMR COSY spectrum showing assignments of (VPGVG)120 at 5 C in a salt- free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3.

48

1 o Figure 3.6. H NMR TOCSY spectrum showing assignments of (VPGVG)120 at 5 C in a salt- free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3.

49

1 13 o Figure 3.7. H and C NMR HSCQ spectrum showing assignments of (VPGVG)120 at 5 C in a salt-free aqueous solution. The spectrum was externally referenced to DSS. The numbering at the proton resonances corresponds to atomic positions in the VPGVG pentapeptide in Figure 3.3. f1 on the right axis corresponds to the carbon chemical shit and f2 on the bottom axis corresponds to the proton chemical shift.

50

Figure 3.8. Chemical shift change as a function of concentration of three salts for (A) the α- protons of glycine, (B) the methyl protons of valine, and (C) the water protons.

51

Figure 3.9. Chemical shift change for (A) the amide protons of (VPGVG)120, (B) the amide protons of poly(acrylamide), and (C) the methyl protons of poly(N,N-dimethylacrylamide).

52

Figure 3.10. Residual change after subtraction of the linear portion (c[mM]) in the chemical shift for the protons from (VPGVG)120 in the presence of NaSCN, plotted together with the residual change in the LCST.

53

Table 3.1. Apparent equilibrium dissociation constants abstracted from isotherm fitting of the

LCST and NMR data of (VPGVG)120 in the presence of NaSCN.

54

Figure 3.11. LCST of (VPGVG)120 as a function of pH.

55

(A) (B)

Figure 3.12. Relative chemical shift changes of (VPGVG)120 amide protons as a function of NaSCN. Experiments were performed at (A) 7.7 and (B) at pH 4.0.

56

Figure 3.13. Relative chemical shift changes of (VPGVG)120 amide proton no. 28 superimposed. Red and green points show changes in relative chemical shift as a function of NaSCN concentration with and without pH 4 control, respectively.

57

Figure 3.14. Binding residual from NMR and LCST data for NaI with (VPGVG)120 after the subtraction of the linear salting-out coefficient.

58

CHAPTER IV

SPECIFIC ION EFFECTS ON TRIGLYCINE: REVERSED HOFMEISTER SERIES AND

INFLUENCE OF SIZE ON ION BINDING

Introduction

The classical interpretation of the Hofmeister series treats ions as water structure makers

(kosmotropes) or breakers (chaotropes).22,73,74 Recent experiments however show that ions do influence bulk water molecules and only affect their hydration shell.57 In fact, the chemical make-up of analyte molecules has the dominate influence for the interactions with salt ions.75

Uncharged biomolecules and surfactants follow a direct Hofmeister series for the ability of salts to influence their solubility, aggregation, and self-assembly in aqueous solutions.8 There can however be reversals in the Hofmeister series if the macromolecules contains formal positive charges. As an example, a reversed series for Lysozyme was observed below its isoelectric point and at low ionic strength.76 Here we have carefully investigated the interactions of Hofmeister anions with three triglycine (GGG) analogs by NMR and ATR-

FTIR spectroscopy. We show that when the end groups of the peptide are capped, anion interactions with the backbone are stronger for weakly hydrated anions compared with strongly hydrated anions. This result is consistent with a direct Hofmeister series. In contrast, when the peptide is uncapped, interactions with the positively charged N-terminus lead to a reversed series for anions. Thus, Hofmeister ordering for anions turns out to be a complex effect involving multiple types of interactions. Tuning the balance of charged and uncharged residues on the protein surfaces could possibly be exploited for many biotechnological applications.

59

Materials and Methods

Sample Preparation

Glycylglycylglycine (GGG) was purchased from Alfa Aeasar and used as received (99%,

Ward Hill, MA). The synthesis of N-acetyl-GGG was performed by acetylation of GGG.

Synthesis of N-acetyl-GGG-amide was performed by starting from an N-acetyl-GG precursor.77,78 Specifically, GG was modified by acetylation of its N-terminus, followed by conjugation with glycinamide. The NaSCN, NaI, NaBr, and Na2SO4 employed in the NMR experiments was purchased from Sigma Aldrich (St. Louis, MO), while NaCl came from VWR

(Radnor, PA). All salts were at least 98% pure, and employing higher purity salts did not change the NMR spectra. 18.2 MΩ•cm purified water from a NANO pure Ultrapure Water system (Dubuque, IA) was used to prepare salt and peptide solutions. Stock solutions of salts and peptides were prepared separately at double the desired concentration and mixed together in a 1:1 (volume to volume) ratio to obtain the desired sample concentration. The total peptide concentration in all experiments was 50 mM. More details of the synthesis and characterization of the capped-GGG molecule are provided in the Supporting Information section of previous reports.30

NMR Titrations

All spectra were acquired on a 400 MHz spectrometer equipped with a 5 mm TXI probe

(Bruker, Billerica, MA) at a temperature of 298 K. For chemical shift assignments of the peptides, spectra employed were [1H,1H]-NOSEY (100 ms mixing time for GGG and 250 ms

- 1 1 mixing time for N-Ac-GGG-NH2 and N-Ac-GGG-CO2 ) and [ H, H]-TOCSY (100 ms mixing time),36 as well as [13C,1H]-HSQC and [13C,1H]-HMBC (J-filter range between 5 and 14 Hz).62

60

TopSpin software (Bruker) was used for data processing. For titrations of peptides with salts,

1H NMR spectra were acquired using presaturation or waterGATE for water suppression.37

Sample spectra were externally referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate

(DSS) (Cambridge Isotope Laboratories) in pure D2O (99.9% D, Cambridge Isotope

Laboratories) in NMR tubes adapted with coaxial inserts (Wilmad-LabGlass). The DSS was always in the inner of the concentric tubes, while the peptide sample was in the outer tube. The

DSS reference was never exposed to the peptide or varying salt concentrations.

Results

GGG Peptides Binding Sites Probed by NMR

The chemical shifts from NMR experiments of the three backbone methylene units of capped triglycine were monitored as a function of salt concentration for five sodium salts:

Na2SO4, NaCl, NaBr, NaI, and NaSCN (Figure 4.1). The top plot in each vertical pair of diagrams represents the total chemical shift observed as a function of salt concentration.

These data can be fit to the following equation:

 [M ]   c[M ] max Kd [M ]

The lower plot shows the data after the linear portion of the data has been subtracted. As

- - can be seen, the data fit very well to a binding isotherm for I and SCN at the CH2 group next to the capped N-terminus (-proton 1) and for SCN- at the middle glycine (-proton 2); the three Kd values are provided in Table 4.1. These interactions for the anions with the peptide are in agreement with those observed by MD simulations.30 No appreciable binding was

61 observed with any of the anions at the -proton 3, which was close to the C-terminus.

Moreover, only linear behavior was observed for Cl- and Br- at all the probed sites. There were,

2- however, nonlinear changes associated with the chemical shifts of SO4 at all three positions

(Figure 4.1). While these latter curves exhibit non-linear behavior, they show opposite curvature from those of the weakly hydrated anions. As such, the curvature may not originate from direct ion-peptide interactions, or even from changes in the peptide secondary structure.

Indeed, infrared data of the amide bands for capped GGG in D2O with 1 M Na2SO4 show no peak shifts or intensity changes within experimental error compared with the peptide in pure

30 2- D2O (ref. 30). Therefore, neither FTIR nor MD data support the idea that SO4 binds or

2- causes substantial changes to the secondary structure. Instead, the NMR curves with SO4 may reflect the anion’s repulsion from the peptide’s immediate solvation shell. Such exclusion

2- could lead to water restructuring in the region between the peptide and SO4 and, consequently, to the non-linear NMR shifts that we observed. It also may be responsible for the opposite curvature seen in the NMR data compared with I- and SCN-. Thus, the results from Figure 4.1 should be consistent with a direct Hofmeister series. Namely, only the most weakly hydrated ions, SCN- and I-, bind to the uncharged glycine residues while the other anions do not.

In a second set of experiments, analogous chemical shifts as above were monitored with uncapped triglycine molecules (Figure 4.2). The chemical shifts varied with salt concentration for all three methylene units. However, there were remarkable differences between the individual -protons. For the methylene group adjacent to the positively charged N-terminus, all the chemical shifts varied in a non-linear fashion (Figure 4.2, -proton 1, top). When the linear part was subtracted out, residual binding isotherms were revealed in the presence of all

2- five sodium salts (Figure 4.2, -proton 1, bottom). In this case, SO4 displayed the tightest Kd

62 value, while I- displayed the weakest (Table 4.1). More specifically, the ion binding ordering was the following:

2- - - - - SO4 > Cl > Br > SCN > I

This is a completely reversed Hofmeister series except for thiocyanate, which again exhibits stronger binding than iodide. By contrast, the chemical shift of the protons associated with the middle methylene unit (-proton 2) and the methylene unit adjacent to the negatively charged C-terminus (-proton 3) showed much less nonlinear variation with added salt for all the monovalent anions. At these two sites, only the -proton 2 data could be fit to a binding isotherm when varying the SCN-concentration and the binding constant in this case was weaker

2- than that found at -proton 1 (Table 4.1). SO4 showed the same type of changes in the chemical shift at -protons 2 and 3 as that found with the capped version of the peptide. Again,

2- this may represent solvent shell reorganization upon depletion of SO4 from the immediate vicinity of the peptide.

Proton NMR titrations of the N-capped GGG peptide with NaSCN and Na2SO4 were also performed. The chemical shift changes of the three backbone methylene protons as well as the

N-terminal methyl protons of the capping group were measured as function of salt concentrations (Figure 4.3). As can be seen, the chemical shift decreased in a non-linear fashion in all cases. Had binding occurred, one would have expected an upward curve for these concentration dependent slopes, rather than the downward curve seen here. In other words, none of the methylene units in the half-capped GGG backbone moieties displayed any apparent anion binding in the presence of NaSCN. This is consistent with the anionic nature of the half

63 capped GGG, which should repel SCN- anions compared with its fully capped and uncapped

2- counterparts. Moreover, SO4 gave rise to even greater downward curve in Figure 4.3 (B), which is consistent with even greater anion repulsion. Such results for the half capped GGG molecule are in agreement with the solubility11,12 and MD simulation30,31 data.

Sodium Sulfate effect on triglycine and end-capped triglycine structure

Chemical shift changes of the -protons (Figures 4.1 and 4.2) show Langmuir isotherm behavior for triglycine and N-acetyltriglycinamide in the presence of Na2SO4, which might suggest direct interactions with the peptide backbone. However, sodium sulfate is known to promote protein folding and might change the structure of the capped and uncapped forms of triglycine or its solvation shells via anion exclusion. To further test these ideas, ATR-FTIR spectra of N-acetyltriglycinamide were measured in D2O and in 1M Na2SO4 in D2O (Figure

4.4). The FTIR spectra show the characteristic amide I band at 1642 cm-1 and a broad shoulder at 1710 cm-1 which is due to the amidation at the C-terminus of the molecule. The amide II band appears as a doublet at 1408 cm-1 and 1477 cm-1 due to mixing between the C-N stretching

-1 40,79,80 and CH3 bending modes. Moreover, the amide III band can be seen at 1338 cm . Overall, these two spectra show identical peak positions and intensities within experimental error.

By contrast with the capped triglycine data, the vibrational spectra of uncapped triglycine in D2O and in 1M Na2SO4in D2O show some subtle spectral differences (Figure 4.5). The amide I band of triglycine appears as a doublet at 1681 cm-1 and 1650 cm-1. The higher frequency peak is from the N-terminus and the lower one is characteristic of the amide I band.81

The absorption peaks at 1593 cm-1 and 1395 cm-1 are the asymmetric and symmetric carboxylate stretches and the peaks at 1482 cm-1 and 1314 cm-1 are the amide II and amide III

64 bands, respectively.40 All these spectral features excluding the 1681 cm-1 band, shift less than

2- -1 -1 2 wavenumbers in the presence of SO4 ions. The 1681 cm shifts approximately 4 cm . The

+ 2- larger shift of this band should be due to ion pairing between NH3 and SO4 . On the other hand, the smaller shifts of the other peaks may be the result of small structural changes in the peptide caused by the ion pairing. Nonetheless, FTIR does not have the spectral resolution to definitively resolve such small changes. These results, especially from the capped triglycine,

2- seem to rule out the idea that SO4 binds directly with uncharged amide moieties. Moreover, thermodynamic data from Von Hippel and coworkers using column chromatography

2- 64 demonstrate that SO4 ions are excluded from amide protons of polyacrylamide gels.

Therefore, the NMR shifts are likely caused by changes in peptide conformation or perhaps its

2- adjacent water structure due to the exclusion of SO4 . To rule out the possibly of a peptide self-association effect, titrations of 1 mM triglycine with Na2SO4 were carried out (Figure 4.6).

The results are mostly identical to the titrations of 50 mM triglycine with Na2SO4 (Figure 4.1 and 4.2).

Discussion and Conclusion

Triglycine and two analogues served as model systems to study the role of electrostatics as well as the influence of molecular size on ion binding. The positively charged N-terminus of the uncapped GGG peptide followed a ‘Reversed’ Hofmeister series with more strongly

2- - - - – hydrated anions, SO4 and Cl , binding tighter than weakly hydrated SCN , I , and Br anions.

This type of binding interaction is electrostatic in nature and should dominate the behavior

(e.g. solubility) of the peptide in aqueous solutions. The backbone α-proton no. 2 of the peptide, however, followed the regular Hofmeister series with only weakly hydrated SCN- ions binding.

65

Capping the charges on both termini of the peptide also resulted in a regular Hofmeister series

2- - - - with SO4 and Cl being repelled and SCN and I both binding with dissociation constants close to 1 M (Figure 4.7).

We found it surprising that these ions showed apparent binding constants that were over two orders of magnitude weaker than for the same type of backbone α-protons of a large elastin-like protein (ELP).29 These results suggest that there is an influence of size on the ability of ions to bind. For all three GGG analogues, SCN- and I- ions were slightly repelled likely due to these small peptides being completely surrounded by water, whereas the large ELP can experience partial dehydration creating low dielectric regions.29,72,82–84 A comprehensive study on the protein-size influence on the affinity for ion binding is currently underway in our lab.

66

Figure 4.1. Relative chemical shift changes of N-Ac-GGG-NH2’s α-protons as a function of salt concentration for five sodium salts.

67

Table 4.1. Fitted values for apparent dissociation constants, Kd, for α-Protons in different aqueous salt solutions with glycylglycylglycine and N-Acetyl-glycylglycylglycinamide. A 95% confidence level was used to extract error bars from the fits. Kd values above 1000 mM represent weaker binding, and blanks were left when no binding could be observed at all within experimental error.

68

Figure 4.2. Relative chemical shift changes of GGG’s α-protons as a function of salt concentration for five sodium salts.

69

Figure 4.3. Relative chemical shift changes of the aliphatic protons of 50 mM N-Ac-GGG- − CO2 (half-capped GGG) as a function of (A) NaSCN concentration and (B) Na2SO4 concentration. The data points are connected with lines as a guide to the eye.

70

1M Na SO 2 4 0.020 D O 2

0.015

0.010

0.005

Absorbance (a.u.)

0.000

1300 1400 1500 1600 1700 1800 -1 Wavenumber (cm )

Figure 4.4. FTIR spectra of 100 mM N-acetyltriglycinamide in D2O (red) and in 1 M Na2SO4 (black).

71

1M Na SO 2 4 0.020 D O 2

0.015

0.010

0.005

Absorbance (a.u.)

0.000

1300 1400 1500 1600 1700 1800 -1 Wavenumber (cm )

Figure 4.5. FTIR spectra of 100 mM triglycine in D2O (red) and 1 M Na2SO4 (black).

72

alpha 2 0.02 alpha 1 alpha 3 0.00

-0.02 -0.04 -0.06 (ppm) -0.08

 -0.10 -0.12

-0.14 0 200 400 600 800 1000 Concentration (mM)

Figure 4.6. Chemical shift changes of the α-protons of 1 mM triglycine as a function of Na2SO4 concentration. Data points are connected to guide the eye.

73

Figure 4.7. A schematic picture of binding sites of weakly hydrated anions (thiocyanate) and strongly hydrated anions (sulfate) at the capped versus uncapped triglycine.

74

CHAPTER V

BEYOND THE HOFMEISTER SERIES AND FUTURE WORK

Perspective. The Hofmeister effect is ubiquitous in physics, chemistry, and biology. As such, its widespread occurrence has attracted the attention of many researchers. The traditional rationalization of the Hofmeister series was based on the ability of ions to cause long range ordering or disordering of bulk water in aqueous solutions. Weakly hydrated ions were called

“chaotropes” presumably because they disrupted water’s bulk hydrogen bond network (chaos).

Strongly hydrated ions were “kosmotropes” because they were assumed to order bulk water molecules at long range (cosmos). These classical interpretations are now known to be inadequate for two reasons: 1) ions have been experimentally and theoretically shown not to affect water molecules outside of their first hydration shell.57,85 2) the classical interpretation ignores the chemical makeup other molecules in the aqueous salt solution.75

The correct interpretation of the Hofmeister series at the molecular level requires the consideration of the interplay of all species present: water, ions, and biomolecules. In fact, many Hofmeister series exist depending on molecular size and charge. As an example, cations do not bind to protein backbones,25 but do bind to negatively charged amino acid side chains.26

Anions bind to both protein backbones29 and positively charged side chains.30,31 In fact, a reversed Hofmeister series is observed when basic amino acid residues are present.76

Moreover, anion binding affinity was shown to be two orders of magnitude weaker for a small molecule compared to a large one. Overall, such molecular level details of the factors that influence cation and anion binding can be exploited for many scientific applications.

75

Exploiting Polar/Nonpolar Hybrid Binding Sites

Weakly hydrated perchlorate anions are toxic and can disrupt thyroid function.86 This is a consequence of the ability of perchlorate to competitively bind tighter to proteins than iodide, which follows a direct Hofmeister series. In recent years, many regions of the U.S. have seen perchlorate concentrations spike as a result explosive manufacturing which consequently leads to contaminated drinking water.87 To protect human health, the EPA currently regulates levels of the amount of perchlorate ion allowed in water sources. Moreover, the limits of detection are in the ppm to ppb range. Therefore, materials that are selective for the detection and/or removal of this ion are needed. We have shown previously that weakly hydrated anions preferentially interact with hybrid polar/nonpolar units on protein backbones.29–31 These findings should aid in the design of new molecules that can be exploited for the engineering of novel materials. As an example, incorporating polar/nonpolar binding site pockets of specific sizes and geometries should allow for selective interactions with specific ions. If precisely designed, interaction sites should provide multivalent binding of anions which could increase the affinity of ions orders of magnitude compared to typical Hofmeister interactions. Along with having the potential to be used as stationary phases in column chromatography, these novel materials could be chemically combined with fluorescent compounds for optical detection. (Figure 5.1)

76

Figure 5.1. Assortment of molecules. Materials containing α-carbon binding sites of polyglycine or quaternary nitrogens of other compounds could potentially be designed and incorporated with chromophores or fluorophores like dansyl chloride.

77

Pharmaceutical Formulations

Many pharmaceutical companies have begun the abandonment of marketing small synthetic organic molecules. Monoclonal antibodies are the next generation of drug molecules being developed for immunotherapy. Recently, we have been contacted by MedImmune, a subsidiary of the pharmaceutical company AstraZeneca, to apply our multi-instrumentation experimental approaches to some of their drug molecules. These monoclonal antibody molecules can aggregate and undergo liquid/liquid phase separations and form aqueous two phase systems (ATPS) under specific conditions. Antibody aggregation in vivo can be toxic. It is therefore important to understand the factors and conditions by which IgGs aggregate. High concentrations of polyethyleneoxide have been shown to influence the aggregation/disaggregation behavior of antibodies in aqueous solutions. 88,89 Salts can also affect antibody behavior in an ion specific fashion and should follow the Hofmeister series.90

The idea is to develop formulations that can preserve the shelf life of the high concentration protein solutions. We therefore plan to apply the conclusions of our work to their systems. In a preliminary set of experiments we followed the effect of NaCl on the disaggregation behavior of a specific IgG drug molecule (MEDI528) at 90 mg/mL in the presence of 30 mg/mL

PEG3350. Light scattering was measured as function of temperature and NaCl concentration

(Figure 5.2). As can be seen increasing concentrations of NaCl decreases the disaggregation temperature of the IgG. The light scattering profiles also show distinct steps in the transition.

There appears to be a shallow slow step followed by a steeper faster step. The molecular mechanisms of these effects will be studied in future experiments.

78

Figure 5.2. Preliminary results for the effect of NaCl on the disaggregation behavior of 90 mg/mL IgG drug molecules (MEDI528) in the presence of 30 mg/mL PEG3350. Light scattering was measured as function of temperature and NaCl concentration.

79

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VITA

Kelvin Blaine Rembert II 104 Chemistry Building The Pennsylvania State University University Park PA 16802 Email: [email protected]

Education

 PhD, Chemistry, Pennsylvania State University, University Park PA, 2014

 BS, Chemistry, University of West Georgia, Carrollton GA, 2009

Publications

 Rembert, K. B.; Okur, H. I.; Hilty, C.; Cremer, P.S. “An NH Moiety is not Required for Anion Binding to Amides in Aqueous Solution.” Submitted 2014  Hladílková, J.; Heyda, J.; Rembert, K. B.; Okur, H. I; Kurra, Y.; Liu, W. R.; Hilty, C.; Jungwirth, P.; Cremer, P. S. “Effects of End Group Termination on Salting-Out Constants for Triglycine.” J. Phys. Chem. Lett. 2013, 4, 4069-4073.  Peterova, J.; Rembert, K. B.; Heyda, J.; Kurra, Y.; Okur, H. I.; Liu, W. R.; Hilty, C.; Jungwirth, P.; Cremer, P. S. “Reversal of the Hofmeister Series: Specific Ion Effects on Peptides.” J. Phys. Chem B 2013, 117, 8150-8158.  Rembert, K. B.; Peterova, J.; Heyda, J.; Hilty, C.; Jungwirth, P.; Cremer, P. S. “The Molecular Mechanisms of Ion-specific Effects on Proteins.” J. Am. Chem. Soc. 2012, 134, 10039-10046. Received coverage in C&E News.  Nguyen, T. T.; Rembert, K.; Conboy, J. C. “Label-Free Detection of Drug-Membrane Association Using Ultraviolet−Visible Sum-Frequency Generation.” J. Am. Chem. Soc. 2009, 131, 1401-1403.

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