Optical Spectroscopies: Absorbance, Circular Dichroism, Fluorescence

Abhi Nath MEDCH 528 1/20/2016 Outline

UV-Vis Absorbance essentials of spectroscopy Beer’s law spectrophotometer and monochromator principles isosbestic points kinetic techniques Circular Dichroism linear vs. circular polarization origins of ellipticity protein CD signals Fluorescence fluorescence vs. phosphorescence steady-state intensity & spectral shifts time-resolved fluorescence fluorescence anisotropy Absorbance Across the EM Spectrum

chemwiki.ucdavis.edu Electronic Spectroscopy excited state

ground state

Jablonski, Nature 131:839 (1933)

λmax S1

FWHM Absorbance

S0 Wavelength Deﬁning Absorbance

transmittance " % I I0 T = A ≈ OD = log10 $ ' # I & I0 absorbance

optical density pathlength

Beer’s Law: A = εbc concentration (aka Beer-Lambert Law) extinction coefﬁcient [conc-1][length-1] Spectrophotometer Schematics

single-beam

dual-beam, dual detector

dual-beam, single detector

adapted from Wikipedia A Simple Monochromator

slit width determines bandpass – resolution vs. sensitivity

chemwiki.ucdavis.edu Intrinsic Chromophores in Proteins RIOCHEMISTRY

6000- I -'-T---1T ~- -1 I I I I ) r -1 cm -1 (M

\ Trp

a 2000- 4 \ i 0 I Tyr

Cystine

~~ 260 270 280 290 300 310 320 0 I WAVELENGTH (rng ) 260 270 280 290 300 310 WAVELENGTH (mu1 20 mM phosphate, pHFIGURE 6.5, 16.0 : Absorption M Gdn HCl curves of N-acetyl-m-tryptophan-Edelhoch, Biochemistry 6:1948 (1967) amide (upper), glycyl-L-tyrosylglycine (middle), and FIGURE 2: Comparison of absorption spectrum of cystine (lower) in 6.0 M guanidine hydrochloride (pH chymotrypsinogen (solid line) (1.61 x lo-$ M) with 6.5b0.02 phosphate buffer. solution of model compounds containing equiresidue concentrations of N-acetyl-m-tryptophanamide, glycyl- L-tyrosylglycine, and cystine in 6.0 M guanidine hydrochloride (pH 6.5E0.02 M phosphate buffer. (insert) most closely resembles the structure of tryptophan in Absorption difference spectrum between protein and proteins. It was ascertained that this compound was pure model solution. by recrystallizing it from acetone and then from ethyl alcohol. No changes in absorption properties were observed after each recrystallization. Its spectrum is shown in Figure 1. In contrast to the tryptophan derivatives, peptide substitution at the a-amino group of tyrosine had very little effect on the absorption properties of the phenolic chromophore. The molar extinction coefficient at the absorption peak was essentially constant in the four ty- rosy1 derivatives listed in Table 11. The compound Gly- L-Tyr-Gly was selected as the most suitable model to represent the behavior of tyrosyl residues in proteins, and its spectrum is shown in Figure 1. The only other a-amino acid found in nonconjugated proteins that absorbs above -270 mp is cystine. How-

TABLE 11: Absorption Properties of Tyrosyl Derivatives in 6.0 M Guanidine Hydrochloride (pH 6.5).

Peak 1 (mp) e OL I I I I I 260 270 280 290 300 310 GI y-L-Tyr-Gly 275.5 1470. WAVELENGTH (mpl Acetyl+ tyrosinamide 275.5 1490 FIGURE 3: Comparison of absorption spectrum of tryp- N-Acetyl-L-tyrosyl ethyl ester 275.5 1500. sinogen (2.50 X 10-6 M) (solid line) with solution of Tyrosine model compounds containing equiresidue concentra- Fisher 275.5 147F tions of N-acetyl-DL-tryptophanamide, glycyl-L- Schwarz 275.5 1515 tyrosylglycine, and cystine in 6.0 M guanidine hydro- L-Leucyl-L-tyrosine 275.5 1500 chloride (pH 6.5k0.02 M phosphate buffer. (insert)

a Solutions contained 0.02 M phosphate. Absorption difference spectrum between protein and 1950 model solution.

FDFLHOCH Extrinsic Chromophores: Substrates/Products

ChemWiki, chemwiki.ucdavis.edu Soderberg, Organic Chemistry With a Biological Emphasis Extrinsic Chromophores:Prosthetic Groups

Das et al., Anal. Chem. 81:3754 (2009) Figure 1. (A) Schematic representation of CYP3A4-Nanodisc immobilized Ag nanobiosensor fabricated using nanosphere lithography (NSL), following by the binding of a drug molecule to the protein immobilized on the surface. (B) Solution UV-Vis absorption spectra of CYP3A4-Nanodisc in the following states: (1) low spin substrate free ferric state with a Soret band at 415 nm (blue solid line), (2) high spin type I drug bound ferric state with aSoretbandat391nm(reddottedline),and(3)lowspintypeIIdrugboundferricstatewithSoretbandat425nm(greendashedline).Theinset shows the detailed changes in the Q-band region. (C) Schematic notations of 11-MUA, the drug and CYP3A4-Nanodiscs. CYP3A4 is shown as a blue space filled model inserted into Nanodiscs. Nanodiscs are nanometer sized discoidal particles with two membrane scaffold proteins (MSP) shown in red ribbon wrapped around a lipid bilayer (shown with the lipid headgroups in red, and the lipid tails in green) in a belt-like manner. quantities of protein would be an important contribution to the initial Rosa, CA); and POPC (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phospho- drug discovery process. choline) from Avanti Polar Lipids Inc. (Alabaster, AL). In the work presented here, we develop a nanoparticle-based Expression and Purification of CYP3A4 Nanodiscs. Cy- sensor prototype for detecting drug binding to membrane proteins tochrome P450 3A4 (CYP3A4) with a C-terminal histidine tag was in a supported lipid bilayer system. Different types of drugs when expressed from the NF-14 construct in the PCWori+ vector as bound to human membrane CYP3A4 in Nanodiscs induce different previously described.34,39,42-44 The CYP3A4 in NF-14 pCWOri+ shifts in the optical absorption spectra of the heme prosthetic vector was a generous gift from Dr. F. P. Guengerich (Vanderbilt group. Herein, we demonstrate that these binding events can be University, Nashville, TN). CYP3A4 was expressed and purified from detected with high sensitivity using localized surface plasmon Escherichia coli as previously described with minor modifications.34 resonance spectroscopy. Human CYP3A4 was assembled into Nanodiscs using the membrane scaffold protein MSP1D1(-),45 in which the poly(histidine) MATERIALS AND METHODS tag is removed.34 Briefly, purified CYP3A4 (in 0.1% Emulgen 913) Materials. Silver shot was purchased from Alfa Aesar (#11357 from the E. coli expression system was mixed with the disk 1-3mmdiameter,Premion,99.9999%).Tungstenvapordeposition reconstitution mixture containing MSP1D1(-), POPC, and sodium boats were acquired from R.D. Mathis (Long Beach, CA). cholate present in 1:65:130 molar ratios. The ratio of CYP3A4/ Polystyrene nanospheres with diameters of 280 ± 4 nm were MSP1D1(-) was kept at 1:10. The detergents (cholate and Emulgen) received as a suspension in water (Interfacial Dynamics Corpora- were removed by treatment with Amberlite (XAD-2), to initiate the tion, Portland) and were used without further treatment. The self-assembly of Nanodiscs. The resultant mixture was then purified buffer salts (KH2PO4 · 3H2O and KH2PO4) and fisherbrand No. using Ni-NTA column to remove the empty Nanodiscs followed by 2 glass coverslips with 18 mm diameter were obtained from size exclusion chromatography to obtain homogeneous CYP3A4- Fisher Scientific (Pittsburgh, PA). For all steps of substrate Nanodiscs in 100 mM potassium phosphate buffer (pH 7.4). preparation, water purified to a resistivity of 18.2 MΩ · cm with The result of this self-assembly reaction is monomeric CYP3A4 Millipore cartridges (Marlborough, MA) was used. incorporated into a 10 nm discoidal POPC bilayer stabilized by 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride the encircling amphipathic membrane scaffold protein belt. The (EDC) was purchased from Pierce (Rockford, IL). Sodium cholate, CYP3A4-Nanodiscs were prepared in a substrate-free form and 11-mercaptoundecanoic acid (11-MUA), bromocriptine (BC), eryth- romycin (ERY), testosterone (TST), lovastatin (LVS), androstenedi- (42) Domanski, T. L.; Liu, J.; Harlow, G. R.; Halpert, J. R. Arch. Biochem. Biophys. 1998, 350, 223–232. one (ADS), alpha-naphthoflavone (ANF), nifedipine (NFD), keto- (43) Hosea, N. A.; Miller, G. P.; Guengerich, F. P. Biochemistry 2000, 39,5929– conazole (KTC), itraconazole (ITZ), tranylcypromine (TCA), diclofenac 5939. (DCF), terfenadine (TRF), and Amberlite (XAD-2) were purchased (44) Gillam, E. M. J.; Baba, T.; Kim, B. R.; Ohmori, S.; Guengerich, F. P. Arch. Biochem. Biophys. 1993, 305, 123–131. from Sigma-Aldrich-Fluka. CHAPS is from Anatrace, Inc. (Maumee, (45) Denisov, I. G.; Grinkova, Y. V.; Lazarides, A. A.; Sligar, S. G. J. Am. Chem. OH), Emulgen 913 from Karlan Research Products Corp. (Santa Soc. 2004, 126, 3477–3487.

3756 Analytical Chemistry, Vol. 81, No. 10, May 15, 2009 Spectral Titrations 4202 Biochemistry, Vol. 45,1-pyrenebutanol No. 13, 2006 (type I ligand) Fernando et al. binding CYP3A4

“isosbestic” point consistent with 2-state system Fernando et al. Biochemistry 45:4199 (2006)

FIGURE 2: Interactions of CYP3A4 with PMA monitored by the substrate-induced type-II spectral transition. (a) A series of the absorbance spectra obtained at no substrate present and at 0.42, 1.3, 1.7, 4.2 10.4, 12.4, 24.7, 30.8, and 48.9 µM PMA. The inset shows the differential spectra obtained by subtraction of the first FIGURE 1: Interactions of CYP3A4 with 1-PB monitored by the spectrum of the series (at no substrate present). (b) The same data substrate-induced spin shift. (a) A series of the absorbance spectra shown as the fraction of the (water-ligated) low-spin state P450 obtained at no substrate present and at 0.78, 1.6, 3.1, 5.5, 7.8, 10.1, versus the concentration of the substrate. The solid line shows the 11.7, 17.2, 25.7, and 38.0 µM1-PB.Theinsetshowsthedifferential approximation of this data set with a binary association equation spectra obtained by subtraction of the first spectrum of the series with KD ) 7.6 µM and the maximal amplitude of the changes in (at no substrate present). (b) The same data shown as the plot of the content of the P450 low-spin state of 58%. The concentration the percent of the high-spin P450 versus the concentration of the of the enzyme was 1.2 µM. Other conditions as indicated in Figure substrate. The line shows the approximation of this data set by the 1. Hill equation with S50 ) 8.6 µM, n ) 1.8, and the maximal amplitude of the spin shift of 48%. The reaction mixture contained 1.45 µM CYP3A4 in 0.1 M Na-HEPES buffer, pH 7.4, 1 mM substrate are mixed at a 1:1 molar ratio, the method is DTT, 1 mM EDTA and was kept at 25 °C. selective for the formation of a binary complex with substrate bound at the higher affinity site. the ligand sphere of the heme iron (Figure 2a). This type of interaction is associated with a displacement of the Soret Changes in the specific fluorescence of 1-PB upon dilution band of the low-spin heme protein from 418 to 426-427 of a 1:1 mixture with CYP3A4 are shown in Figure 3a. The nm (see Figure 2a, inset). The titration curve in Figure 2b signal was normalized to the protein concentration and presents these changes in terms of a decrease in the fraction corrected for the internal filter effect as described in of the “normal” (water-ligated) low-spin state of the enzyme Experimental Procedures, so that in the absence of FRET and obeys the equation for the equilibrium of bimolecular the spectra are not expected to be affected by the concentra- association (eq 1) with a dissociation constant (KD) of 7.7 tion of the mixture. As shown in the inset in Figure 3a, the ( 2.3 µM, as obtained by averaging the results of 3 principal component analysis applied to these series of individual experiments (Table 1). spectra yields the first principal component signifying a Interactions of CYP3A4 with 1-PB, PMA, and Bromocrip- concomitant decrease of all emission bands of 1-PB upon tine Monitored by FRET to the Heme of the Enzyme. As the its interaction with CYP3A4. As shown in the inset in Figure interactions of pyrenes with CYP3A4 are expected to result 3a, the second principal component deduced from this in FRET from the substrate to the heme (21, 22, 32), we analysis may reveal some minor changes in the degree of monitored the changes in fluorescence of 1-PB and 1-PMA excimerization of 1-PB during the experiment. However, the upon dilution of the enzyme-substrate mixtures. Thus, a amplitude of this component was negligible, and it was not series of emission spectra were taken at a constant molar considered in further analysis. Although the decrease in the ratio of enzyme:substrate and simultaneously decreasing intensity of fluorescence of 1-PB observed here may result concentrations of both compounds. In the case of multisite from several different reasons, FRET appears to be the most binding, this approach allows us to consider the binding plausible cause. The principal component analysis did not events separately, provided that the affinities of the two sites reveal any significant changes in the shape of the emission remain considerably different irrespective of substrate satura- spectra (Figure 3a,b, insets), which would be anticipated if tion of the high-affinity site. When the enzyme and the the changes in the intensity of fluorescence were caused by Non-2-State Systems:

993 RhodopsinSATOKI. KAWAMURA, Photobleaching FUMIO TOKUNAGA and TAM Yostu/.I\M-.ji

no isosbestic point

measured ot-SOY

Wovelength, nm 0.4- (b) Kawamura et al. Vision Res. 17:991 (1977)

0.1.. Suspension measured at -72OC _.

Wovelength, nm 1.0

z 0.6

6 D

$ $ .o .a

Retina Cl.2 ineosured ot-72°C

C 340 400 500 600 700 Wovelength, “m Fig. 2. Formation of metarhodopsin I by irradiation of bullfrog rhodopsins in the three different preparations. (a) An extract, irradiated at -50°C with light longer than 560nm successively for a total of 2, 4, 8, 16, 32, 64, 128 and 192 min (curves 2-9 respectively). (b) A suspension, irradiated at -72°C with light longer than 560nm successively for a total of 1, 2, 4, 8, 16 and 32min (curves 2-7 respectively). (c) A retina, irradiated at -72°C with tight longer than S6Onm successively for a total of 1, 2, 4, 8, 16, 32, 64, 128 and 192rnin (curves 2-10 respectively). pension lies at almost the same wavelength as in the between the extract and the suspension. (1) Rhodop- retina, but at longer wavelength than in the extract; sin or its bleaching intermediate differs in the confor- metarhodopsin I is an exception (Table 1). mation of chromophore between the suspension and the extract. (2) The absorption spectrum of rhodopsin DISCUSSION or an intermediate is slightly affected by the environment; i.e. the electronic states of the chromophore Absorption spectra in the extract and in the suspmsion in the digitonin micelle (extract) may be somewhat Two possible explanations may be given for the different from those in the phospholipid biiayer (sus- inconsistency in the spectra of the intermediates pension). Transient Absorbance: Pump-Probe Spectroscopy

(30 cm ≈ 1 ns)

Menon et al. Physiol. Rev. (2001) 81:1659 Stopped-Flow Spectroscopy Summary of UV-Vis Absorbance convenient probe of concentration label-free steady-state & kinetic insight can interrogate very short timescales

Caveats low resolution intrinsic chromophores not very environment-sensitive Outline

Wikipedia Circularly Polarized Light (CPL)

Wikipedia Linear vs. Circular vs. Elliptical Polarizations

differential absorption Wikipedia Measuring Circular Dichroism

∆absorbance: ΔA = AL − AR ΔA molar circular dichroism: Δε = bc tanθ 1 $ I − I ' molar ellipticity: θ ≈ = R L = 3298Δε (in °cm2/dmol) [ ] & ) bc bc % IR + IL (

commonly reported as mean residue ellipticity: molar ellipticity divided by # of amino acids in protein chemwiki.ucdavis.edu Optically Active Molecules chiral molecules interact differentially with circularly polarized light differential refraction: optical rotation differential absorbance: circular dichroism

(any asymmetric single molecule is optically active, even without a chiral center) 166 WY.B. Gratzer (Discussion Meeting) effect near 200nm. A small positive Cotton effect, centred at about 218nm also appeared to be present (Holzwarth & Doty I965). These results have been sub- stantially confirmed by recent work of Velluz & Legrand (I965), using probably the most advanced instrumentation at present available (figure 3(a)). The rotational strengths of the two Cotton effects are respectively about 17 and 2 x 10-40 c.g.s. units. (A further very weak Cotton effect at about 243 nm seen in the results of VelluzFar-UV & Legrand CD of on Proteinpolyglutamic 2’ Structureacid most probably represents a contribution from the y carboxylate groups.) The same workershave also examined a tripeptide, glycyl-L-lysyl-L-glutamic acid, and at a pH at which the acand y carboxyl groups

6 RC α-helix β-sheet 8 (a) (b)() 6 CD absorbance

2 - C~~~~~~~~~~~~~~~~4- t j 9/

-3 JIt--I1 I I I I r-I I 190 200 220 240 190 200 220 240 190 200 220 240 A(nm) FIGURE 3. Circular dichroism and absorption spectra (broken lines) of the polypeptide chain (a) in the random coil state; (b) a helix; (c) flconformation. (Molar ellipticites are in deg cm2/ decimole; absorption bands are plotted in terms of mean molar residue absorptivity.) Data are compiled from sources mentioned in the text, and are based on poly-L-glutamic acid and puly-L-lysine in aqueous solution. Gratzer, P. Roy. Soc. Lond. A. 297:163 (1967) are protonated, a circulardichroism curve similar to that of a random polypeptide is observed. The maximum of the main Cotton effect, however, lies at about 192nm, rather than 198nm, as in the polymer, and is therefore much nearer the maximum of the absorption band. The reason for the displacement of the Cotton effect maximum in the randomly coiled polymer from the absorption maximum of the peptide band is not readily explicable. The possibility that ionized polyglutamic acid is not truely random may be considered. It is clearly desirable that other disorderedpolypeptides should be examined.

OPTICAL ACTIVITY OF THE a HELIX It has been known since the work of Doty & Yang (I956) that the optical rotatory dispersion of achelical polypeptides in solution does not obey a simple Drude law. The theoretical treatment by Moffittof the optical rotatory dispersionfar from the optically active absorption bands, in terms of two Drude-type terms, one for each of the two exciton components of the 1T -- 7f* absorption band, led to the celebrated equation (Moffitt & Yang I956)

[m']A-ao (A2?A ) +?b ( )

This content downloaded from 173.250.196.104 on Mon, 20 Jan 2014 22:19:22 PM All use subject to JSTOR Terms and Conditions Biochemistry: Pritsker et al. Proc. Natl. Acad. Sci. USA 95 (1998) 7289

2 1͞2 Ez ϭ (2 cos ␪ sin ␪)͞{[(1 Ϫ n21 )

2 2 2 1͞2 ϫ(1 ϩ n21 )sin␪ Ϫ n21 ] }, where ␪ is the angle of incidence between the light beam and the prism normal (45°) and n21 is the reflective index of the germanium (taken to be 4.03) divided by the reflective index of the membrane (taken to be 1.5). Under these conditions, Ex, Ey,andEz are 1.40, 1.52, and 1.64, respectively. The electric field components together with the dichroic ratio [defined as the ratio between absorption of parallel (A࿣)andperpendic- ATR ular (AЌ)polarizedlight,R ϭ A࿣͞AЌ]areusedtocalculate the orientation order parameter, ᒃ,bythefollowingformula:

2 2 2 2 2 R ϭ (Ex ͞Ey ) ϩ (Ez ͞Ey ){ᒃcos ␣ ϩ (1 Ϫ ᒃ)͞3}͞{1͞2(ᒃsin2␣) ϩ (1 Ϫ ᒃ)͞3}.

Lipid order parameters were obtained from the lipid symmet- ric (2,852 cmϪ1)andasymmetric(2,924cmϪ1)stretchingmode by using ␣ ϭ 90° (44). Determining the Oligomeric States of the Peptides by SDS͞PAGE. The experiments were done as described (17, 45). The peptides were dissolved in a buffer composed of 0.065M Tris⅐HCl (pH 6.8), 2% SDS, and 10% glycerol by sonication. FIG.1. DosedependenceoflipidmixingofPOPGLUVinduced Fixing, staining, and destaining times were shorted to 1 min, by WT and WT-D peptides. Peptide aliquots were added to mixtures 1hr,andovernight,respectively,todecreasediffusioneffects. of LUV (22 ␮Mphospholipidconcentration)containing0.6%(molar Resonance Energy Transfer Measurements. Resonance en- ratio) each of NBD-PE and Rho-PE and unlabeled LUV (88 ␮M ergy transfer experiments were performed by using NBD- phospholipid concentration) in PBS. The increase of the fluorescence labeled peptides serving as energy donors and Rho-labeled intensity of NBD-PE was measured at 10 min after the addition of the peptide, and the percentage from maximum was plotted versus the peptides serving as energy acceptors as described in details peptide͞lipid molar ratio. The fluorescence intensity on the addition elsewhere (46, 17). of Triton X-100 (Triton Biosciences) (0.25% vol͞vol) was referred to as 100%. Squares, WT; triangles, WT-D. (Inset)Timeprofileofthe RESULTS lipid mixing at peptide͞lipid ratio of 0.075. ApeptiderepresentingtheN-terminal33-aasegmentofgp41 ␤-sheet, respectively, whereas at 80:1 lipid͞peptide ratio, WT of HIV-1 (LAV1a), WT, and its all [smcap]d-amino acid and WT-D have 25 and 26% ␣-helix and 46 and 50% ␤-sheet, enantiomer, WT-D, were synthesized and were labeled fluo- respectively. There is an increase in ␣-helix͞␤-sheet ratio with rescently at their N-terminal amino acid with either NBD (an an increase in the lipid͞peptide ratio [which causes an increase environmentally sensitive probe and an energy donor) or Rho in the fraction of membrane inserted peptide (17)], as has been (an energy acceptor). The sequence of the WT peptide is: shown also with short versions of HIV-1 fusion peptide (49). Incorporation of the Peptides into Bilayers. The fluores- AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL cence emission intensity of the NBD-labeled peptides was Liposome Fusion Induced by WT and WT-D. In separate monitored in PBS alone or in the presence of POPG-SUV. In CD Sign Depends on Stereochemistry␮ experiments, increasing amounts of each peptide were added the buffer, the peptides (0.1 M) exhibited emission spectra to a fixed amount of vesicles, and the maximal values of the fluorescence intensity, reached after the addition of the peptides, were plotted as a function of the peptide͞lipid molar D-amino acids ratio (Fig. 1). The data reveal that WT and WT-D have similar potency and kinetics in inducing lipid mixing. Secondary Structure of the Peptides in trifluoroethanol͞ Water. The ␣-helical content of WT in 40% trifluoroethanol͞ water was found to be 48% (Fig. 2A). WT-D showed spectra characteristic of all D-amino acids containing ␣-helix peptides with the mirror ellipticity at 222 corresponding to 47% ␣-helical content (Fig. 2A)similarlytootherenantiomericpeptides (23, 24, 47). Secondary Structure of the Peptides in Lipid Multibilayers Determined by ATR-FTIR Spectroscopy. The amide I region between 1,648 and 1,660 cmϪ1 is characteristic of ␣-helical L-amino acids structure (48). A spectrum of the amide I region for WT and WT-D bound to dry POPG multibilayers is shown in Fig. 2B. The contributions of the various secondary structure elements to the amide I peak were obtained by curve fitting by using FIG.2. (A)CDspectraofWT(solidline)andWT-D(dashedline) PEAKFIT (Jandel Scientific, San Rafael, CA) and the values Ϫ1 peptides in 40% trifluoroethanol.Pritzker (B et)ATR-FTIRspectraofthe al. PNAS 95:7287 (1998) from ref. 48. Two main peaks, located at 1,660 and 1,627 cm , peptides in POPG multibilayers. The samples were prepared as represent ␣-helix and ␤-sheet, respectively. The ␣-helical and described in Material and Methods;peptide͞lipid molar ratio was 1:80. the ␤-sheet contents of WT and WT-D peptides were calcu- The spectra were analyzed by using the curve fitting of the amide I lated at two lipid͞peptide ratios. At 50:1 lipid͞peptide ratio, band area assuming Voight line shapes for the IR peaks. Solid line, WT and WT-D have 18 and 19% ␣-helix and 58 and 56% WT; dashed line, WT-D. Measuring Protein Folding by CD

CORRECTING SPECTRA USING SINGULAR VALUE DECOMPOSITION 59

matrix, A, can be decomposed into three matrices, U, S, and VT.

A USVT Å If the spectra are column vectors in the matrix A, the isosbestic aka isodichroic point U matrix consists of orthonormal basis vectors for the data space. S is a matrix containing a set of numbers called singular values, which weigh the significance of each basis vector in matrix U. VT contains the orthonormal coefficients that fit matrix US to the data matrix A. We analyzed our data matrix A, which contains all the CD spectra of 19 peptides in all different methanol/ buffer solutions (0 to 95% with a 5% increment), for the matrices U, S, and VT. The analysis gave us two large singular values in the S matrix. This means that there are only two significant basis vectors in the data space, supporting the observation of two dominant spe- cies in the transition for all peptides.

Contribution from Aromatic Side Chains FIG. 1. The typical CD spectra of a Y-(VAXAK)3 peptide measured in various methanol/buffer concentrations. Here X is substituted Researchers often observe the CD bands arising from with alanine. The spectKrittanaira indicate t w&o dJohnson.ominant con foAnal.rmation Biochems of absor.b 253:57ing amin (1997)o acid side chains around 280 nm in the random coil and a helix for all peptides. The corrected spectra for near-UV region. These CD bands are usually recog- alanine (—) are identical to the original spectra (---), indicating no nized as an aromatic band resulting from Trp, Tyr, or contribution from its side chain. Phe. These absorbing side chains also contribute to the CD in the far-UV region as has been reported by several laboratories (10 – 12, 27 – 29). the peptides in the same cell used for the CD measurement.

RESULTS AND DISCUSSION CD Measurement on the Peptides The CD spectra of the 20 peptides in a series of sol- vents consisting of methanol and 2 mM phosphate buffer, pH 5.5, show a transition between a random coil and an a helix. All peptides (except the one with proline substitution which is not included in the analysis) adopted the random-coil conformation in buffer and became more helical when the methanol content was increased, as shown for the alanine-substituted peptide in Fig. 1. The CD spectra show a negative band at about 198 nm for the random coil, two negative bands at 222 and 208 nm, and an intense positive band at about 193 nm for the helix. The isodichroic point is located at about 203 nm, indicating two dominant states, the random coil and a helix.

Analysis by Singular Value Decomposition Singular value decomposition is a mathematical theorem that can be applied to spectral analysis (32 – FIG. 2. The corrected (—) and original (---) CD spectra for Y-(VA-

35). The theorem states that a data set written as a WAK)3 .

AID AB 2366 / 6m4c$$$162 10-06-97 14:29:29 aba PROTOCOL Measuring Protein Folding by CD

Reversiblesingle-wavelength Folding of N-terminally Truncated MRE PrP spectral deconvolution 617 abcd3 5 12 intrinsically1.0 stable folding unit PrP(121 to 231) 8

0 –1 –1 –1 0 is not in accordance with the proposed three- 4 –3 C dimensional0.8 structure of the PrP segment 108-218 0 dmol

dmol dmol (Huang et al., 1994) as it lacks the ﬁrst helix 2 2 –6 2 –5 (residues 109 to 122) of the predicted four-helix –4 0.6 –9 bundle. However, several lines of evidence suggest –8 –10 that the main and possibly the only part of PrPC –12 with deﬁned three-dimensional structure is rep- –12 deg cm deg cm deg cm 0.4 resented by residues 121 to 231, and that most of the –16 –3 –3 –15 –3 –15 proposed structural changes linked to the transition C Sc –20

0.2 10

10 10 from PrP to PrP may occur within this segment. Fraction each curve –18 × × × (1) Amino- or carboxy-terminal secondary structure –24 ] ] ] –20 θ θ θ

elements being part of a single, small protein [ [ –21 [ 0.0 –28 0 domain generally cannot be deleted without loss of 0 –24 –25 protein stability and cooperativity of folding, since –32 210 220 230 240 250 260 210 220 230 240 250 260 one-domain0 10 modules 20 appear 30 to 40 fold in 50 a concerted, 60 210 220 230 240 250 260 Wavelength, nm Wavelength, nm cooperative mechanismTemperature, and not ° inC a hierarchical Wavelength, nm process (de Prat Gay et al., 1995a,b). (2) It was demonstrated that peptide fragments comprising isolated helices of myohemerythrin, a four-helix Hornemann & Glockshuber. J. Mol. Biol. 262:614 (1996)bundle protein,Greenﬁeld. spontaneously Nat. Prot. form 1:2527 helical struc-(2006) eftures (Dyson et al., 1992). However, a synthetic 1.0 Figure 4 | Example of the analysis of set of spectra using the CCA. –1 peptide0 spanning the ﬁrst, predicted a-helix s (a,d) Spectra of a model protein containing the C-terminal domain of (residues 106 to 126) did not exhibit a-helical 0.8 structure in aqueous solution and appeared to be a dmol rat striated muscle tropomyosin (CTD) and a fragment containing the 2 mixture of b-sheet and random coil structure (de 0.6 40 Gioia–10 et al., 1994). (3) Proteolytic cleavage of a mutation of a glutamine to leucine (CTDQ263L) collected as a function protein domain should occur mainly at exposed loop regions rather than within secondary structure deg cm 0.4 of temperature, respectively. (b,e) The data in a and d deconvoluted into elements (Price & Johnson, 1993). The experimental 25 –3 evidence–20 thus indicates that the fold of PrP(121-231) three basis curves using the CCA .(c,f) The fraction of each basis curve is incompatible with the structure previously 10 0.2 C Fraction each curve × proposed for PrP . ]

contributing to each spectrum at each temperature. The fractional weights θ It was recently shown that transgenic mice [ natureprotocol Figure 4. Reversible unfolding ( ) and refolding ( ) 0.0 / w W exclusively–30 expressing a PrP variant lacking of the two curves corresponding to the unfoldedof protein PrP(121-231) are in the summed presence of in guanidiniumc chloride residues 32 to 80 are still susceptible to infection m at pH 7.0 and 22°C. a, mean residue ellipticities at by mouse210 PrPSc 220and 230 capable 240 of propagating 250 260 the 0 20 40 60 80 o because they were almost identical. The protein samples222 nm. b, normalized were 1.67 transition.mM The in continuous lines infectious agent, which means that all residues c

. Wavelength, nm result from fitting the data according to a two-state required for the conversion to PrPSc are located Temperature, °C e 100 mM NaCl, 10 mM sodium phosphate buffer, pHtransition. 6.5. Native Data or were GdmCl-denatured collected PrP(121-231) was r diluted 1:11 by 20 mM sodium phosphate (pH 7.0) within segment 81-231 (Fischer et al., 1996). u containing different GdmCl concentrations (final concen- PrP(121-231) represents 74% of this segment and t at 5-deg increments with 2 min equilibration at each point in cells with path lengths of 1 cm. CTD, model protein containing residues 251–284 of rat tration of PrP(121-231): 38 M). After incubation for contains ten of the 13 codons in human PrP, where a m 36 hours at 22 C, the mean residue ellipticity at 222 nm point mutations are assumed to be associated with n striated tropomyosin with GCG at the N terminus. CTDQ263L,° the same peptide with the mutation Q263L. The peptides are cross-linked by disulfide bonds. . was recorded for three minutes in 0.1 cm cuvettes and inherited prion diseases (Scha¨tzl et al., 1995; w averaged. The data were evaluated by a six-parameter fit Prusiner, 1993). The polymorphism at residue 129 and normalized as described (Santoro & Bolen, 1988). w in human PrP, which appears to influence

w susceptibility to the Creutzfeldt-Jakob disease /

/ (Palmer et al., 1991), also lies within PrP(121-231). : Spectra collected as a function of wavelengthprotein and difference in accessible surface area Therefore, it will be most interesting to see whether p t between the unfolded and folded state (Myers et al., transgenic mice exclusively expressing the au- t Analysis of spectra collected as a function1995), has of a temperaturevalue of 8.6 (20.5) kJ mol show−1 M−1 GdmCl the usetonomously of the folding CCA fragment to PrP(121-231) deconvolute will still the spectra to obtain the h

and is in the range expected for a 13.3 kDa protein be susceptible to scrapie. minimum number of basis spectra needed(Myers toet al ﬁt., 1995). the data. Figure 4a andThed reversibilityillustrate of PrP(121-231) the spectra folding raises of the CTD model used here and p We have shown that the amino-terminally principle questions on the folding pathways of PrPC u truncated segment of the mouse prion protein and PrPSc. When we neglect an autocatalytic o the same model protein with the mutation Q263L (ref. 40) (the numbering is that of intact tropomyosin)Sc determined as a

r (residues 121 to 231) is an isolated domain with mechanism for formation of monomeric PrP and tertiary structure and high intrinsic stability, whose simply assume that formation of insoluble, G function of temperature, respectively. The CD spectra suggest that the wild-type CTDSc modelSc protein at 10 C is 60–70% helical, folding and solubility does not require N-glycosyla- protease-resistant PrP -oligomers ((PrP )n ) is an 1

g tion at residues 181 and 198. Most importantly, irreversible process an oligomerization-competent Sc n in agreement with nuclear magnetic resonancerefolding of chemically studies, denatured which PrP(121-231) show is thatPrP monomer it is 73%may be: (1) helical by an on-pathway (63% or (2) of the helices are in a canonical i cooperative and reversible and yields a molecule by an off-pathway folding intermediate of PrPC

h 41 coiled coil and the rest are in linear -helices)indistinguishable and from the the restnative recombinantis disordered(both of which. Replacing could be molten Q263 globule-like; at Safar the coiled coil interface of the s a i protein. We are aware of the fact that the et al., 1994); or (3) the ﬁnal product of PrP folding, l

b CTD with a leucine increases the helical content to B90% (almost all coiled coil) and also increases the stability of the u 40

P molecule . e r Analysis of the change in the spectrum of the wild-type CTD model protein shows that it unfolds in a two-state transition. u t

a When the set of spectra in Figure 4a are deconvoluted into basis spectra using the CCA into three spectra (Fig. 4b), two of the N basis spectra are almost identical, showing that only two conformations are needed to ﬁt the all of the data in Figure 4a. The 6 0

0 percentage of each of the basis curves contributing to the spectra are shown in Figure 4c, where the fraction of each of the two 2 curves corresponding to the unfolded structure are added. The change in the spectrum of the curve corresponding to the folded © form unfolds with a TM of 25 1C (within experimental error of the TM of unfolding when the ellipticity change at 222 nm is followed as a function of temperature). When the set of spectra from the CTD molecule containing the Q263L mutation are deconvoluted, in contrast, at least three curves are needed to fit the data. The helical peptide unfolds to give a spectrum similar to that of the wild-type CTD, but then it begins to aggregate to give a spectrum with some b-sheet characteristics. Two files are provided as supplementary materials. Supplementary Equations has routines in SigmaPlot formats for fitting a variety of unfolding/folding curves for proteins with various numbers of subunits (e.g., monomers, dimers, trimers) to estimate the enthalpy, TM of and heat capacity changes of folding. Supplementary Tutorial has instructions for using DOS versions of the CCA for deconvoluting sets of CD spectra. A copy of the CCA that works in a command window in Windows 95, 98 and XP is also supplied.

Note: Supplementary information is available via the HTML version of this article. Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ ACKNOWLEDGMENTS The work was supported by a National Institutes of Health reprintsandpermissions grant, GM-36326 to N.J.G. and Dr. Sarah E. Hitchcock-DeGregori, and by the Circular Dichroism Facility at Robert Wood Johnson Medical School (UMDNJ). 1. Velluz, L., Legrand, M. & Grosjean, M. Optical Circular Dichroism: Principles, I thank the late Dr. Gerald D. Fasman for supplying copies of programs to Measurements, and Applications (Verlag Chemie Academic Press Inc, New York and deconvolute data sets using the CCA and Dr. Takashi Konno for supplying a London, 1965). SVD program. 2. Beychok, S. Circular dichroism of biological macromolecules. Science 154, 1288–1299 (1966). COMPETING INTERESTS STATEMENT The authors declare that they have no 3. Adler, A.J., Greenﬁeld, N.J. & Fasman, G.D. Circular dichroism and optical rotatory competing ﬁnancial interests. dispersion of proteins and polypeptides. Methods Enzymol. 27, 675–735 (1973).

2534 | VOL.1 NO.6 | 2006 | NATURE PROTOCOLS (3) How kinetic data report on folding in a multidomain protein Where protein domains exist in a multidomain construct, a single domain can be stabilised by its neighbour(s) in one of three ways.

(A) If the neighbour provides favourable interactions in the native state only, then the kinetic barrier to unfolding is raised but the kinetic barrier to folding is unchanged. This results in a change in the chevron plot, as shown in Figure S3. Sidebar: Chevron Plots Notice that the minimum of the chevron plot has shifted to the right, indicating a higher stability.

(2) What is a chevron plot?

7 Isolated Domain Domain in Multidomain Construct 6.5 folds at same rate 6 unfolds slower in multidomain protein 5.5 ) s b o 5 k ln( 4.5

3.5 midpoint shifted to higher [denaturant]

3 0 1 2 3 4 5 6 7 [denaturant] (M)

Figure S2: A sample chevron plot where each black dot represeFntigus ar see paS3:ra tAe kidomnetiaci n folds at the same rate in a multidomain context but unfolds experiment. more slowly. • For unfolding experiments, (those on the red dashed line), a sample of folded protein in buffer is rapidly mixed with an excess of denaturant solution,Bately , Nickson & Clarke HFSP J. (2008) 2:365 (typically 1:10), which results in a high overall concentration of denaturant. The unfolding of the protein is monitored by following changes in a structural probe e.g. CD or intrinsic fluorescence. From these data the unfolding rate constant (ku) can be determined and this is plotted against the concentration of denaturant at which it occurred. • For folding experiments, (those on the blue dashed line), a sample of unfolded protein in high denaturant solution is rapidly mixed with an excess of buffer, resulting in a low overall concentration of denaturant. The same optical probes are used to monitor the protein folding, and the calculated folding rate constant (kf) is again plotted against the concentration of denaturant at which it occurred. • The protein folds more slowly in the presence of denaturant than in pure buffer. Likewise, the higher the concentration of denaturant, the faster the protein unfolds. Thus the resultant plot has a characteristic V-shape – hence it is called a chevron plot. • Provided that there is no change in the structure of the transition state, the logarithm of the folding and unfolding rate constants (lnkf and lnku respectively) will be directly proportional to the concentration of denaturant.

These rate constants are shown as dashed lines in Figure S2, (lnkf in blue, lnku in red), and can be extrapolated back to zero molar denaturant to give the H2O folding rate constant in water (lnkf ) and the unfolding rate constant in water H2O (lnku ). These values can then be used to determine the stability (the free energy of unfolding) of the protein in the absence of denaturant (ΔG = H2O H2O RTln(kf /ku ). • Note that if an intermediate is populated (i.e. the protein is not 2-state) then the chevron plot may be curved. Multiple Binding Sites on c-Myc Oncoprotein ARTICLES

Binding Measured by CD: Disruption of the c-Myc/Max Dimer Figure 3. Circular dichroism (CD) spectra of c-Myc363-381 and c-Myc402-412 upon addition of inhibitors. (a) CD spectra of c-Myc363-381 alone (O), with 10074-G5 (b), and with 10050-C10 (2). (b) CD spectra of c-Myc402-412 alone (O), with 10058-F4 (b), with 10009-G9 (2), with 10075-G5 (9), and with 10031-B8 ([).

monomers

+ inhibitor

heterodimer Figure 4. Disruption of the c-Myc-Max dimer upon addition of 10074-A4: (a) Summation of the two CD spectra: c-Myc and Max(p21) taken independently (O) and CD spectra of the c-Myc-Max(p21) dimer before the addition of 10074-A4 (2) and after the addition of 10074-A4 (b). (b) Competition titration by CD of the c-Myc-Max dimer upon additionHammoudeh of 10074-A4. et al. Error J. Am. bars Chem. represent Soc. 131:7390 SEM. (c) (2009) CD spectra of Max(p22) in the absence (O) and presence (b) of 10074-A4.

Figure 5. CD spectra of c-Myc370-409 upon addition of 10074-A4, showing the induced CD band with a minimum at 245 nm. (a) CD spectra of c-Myc370-409 (O)andc-Myc370-409 after addition of 10074-A4 (b). (b) Titration of the ICD band at 245 nm by serial dilution of a 1:1 mixture of 10074-A4 and c-Myc370-409. Error bars represent SEM. binding in a CD experiment where the extent of c-Myc-Max specific binding to c-Myc (Figure 4c). Information about the dimer formation was monitored by measuring the ellipticity at location of the binding site for 10074-A4 was obtained by 222 nm, indicative of R-helical content (Figure 4b). The monitoring the CD of various truncated c-Myc bHLHZip Max(p21) isoform (which homodimerizes only at very high peptides upon addition of this compound. We monitored the micromolar concentrations)57 was used in this experiment in effect of 10074-A4 on the CD spectra of peptides encompassing order to avoid convoluting the effect on R-helical content by the truncated segments systematically: c-Myc353-405 (Figure S2C formation of Max homodimer. The compound was able to in Supporting Information), c-Myc370-409 (Figure 5a), c- displace Max with an observed Kcomp of 32 ( 3 µM which, Myc380-439 (Figure S2D in Supporting Information), c-Myc390-439 based on the independently determined c-Myc-Max affinity, (Figure S2E in Supporting Information), c-Myc400-439 (Figure corresponds to an inhibitor affinity of 13 ( 3 µM. A similar S2F in Supporting Information), and c-Myc363-381 (Figure S2G experiment performed with Max(p22), an isoform with high in Supporting Information). The spectra of c-Myc370-409 and affinity for homodimer formation, showed that 10074-A4 was c-Myc353-405 show a significant change after the addition of unable to disrupt the Max homodimer, thus confirming its 10074-A4, whereas the CD spectrum of c-Myc353-437 shows a very limited change, retaining its ID structure after the addition (57) Zhang, H.; Fan, S. J.; Prochownik, E. V. J. Biol. Chem. 1997, 272, of 10074-A4 (Figure S2B in Supporting Information). The 17416–17424. spectra are indicative of a binding interaction involving only a

J. AM. CHEM. SOC. 9 VOL. xxx, NO. xx, XXXX G Near-UV CD: Local Structure Around Aromatic Side- Chains B.G. Vertessy et al./FEBS Letters 421 (1998) 83^88 85

86 B.G. Vertessy et al./FEBS Letters 421 (1998) 83^88

Fig. 3. Titration of enzyme with K,L-imido-dUTP. Native (closed symbols) or truncated (open symbols) dUTPase was titrated by stepwise addition of K,L-imido-dUTP. Absolute values of the di¡erential CD signal at 206 nm (closed circles), 260 nm (closed squares) or 270 nm (open 2 0:5 circles) are plotted. Data points were ¢tted (solid lines) to the equation: v3/v[3]=(a3(a 34c1c2) )/2. v3 is the absolute value of the di¡erential ellipticity determined by subtracting the ellipticity of K,L-imido-dUTP and dUTPase solution, measured separately, from the ellipticity measured in their mixture, v[3] is the di¡erential molar ellipticity of the protein-ligand complex, a = c1+c2+Kd;app, c1 and c2 are enzyme and ligand concentrations, respectively). v[3] and Kd;app (apparent dissociation constant) values are given in Table 1. Enzyme concentration for the Fig. 2. Altered binding of K,L-imido-dUTP to native or truncatedfar UV experiment dUTPase (206 nm) as was recorded 12.5 WM, by for nearthe near UV UV CD.experiments A: Spectra(260 or 270 of nm) native was 45 enzymeWM (native alone enzyme) or 40 WM (truncated dUTPase). (closed symbols), K,L-imido-dUTP alone (open symbols) (scattered graphs), and the mixtures (line graphs) of K,L-imido-dUTP with native (solid line) and truncated (dotted line) dUTPase. The spectrum of truncated enzyme is identical to the spectrum of the native enzyme in this wavelength range [13]. B: Di¡erence spectra of native dUTPase4. with DiscussionK,L-imido-dUTP (solidVertessy line) or dUDP et al. (dashed FEBSenzyme line). Lett The activity. dotted 421:83 [13,18]. line In the is(1998) human the dif- enzyme, the C-terminal ference spectrum of truncated dUTPase with K,L-imido-dUTP. Protein and ligand concentration were 45 WM andarm 60 closesWM, upon respectively. the active site when mono-, di- or triphos- The conserved Motif 5 at the £exible C-terminal arm of phates of 2P-deoxyuridine bind to the enzyme in the absence 2 trimeric dUTPases cannot be located in the unliganded crystal of Mg [7]. However, no such movement can be seen when structures of the enzyme from E. coli, Homo sapiens or the dUDP binds to the bacterial enzyme [10]. In the present study argue for an involvement of Phe in the binding of thelentivirus triphos- of feline immunode¢ciencythe di¡erence [7,9,17]. signal Its (Fig. functional 2B, dottedwe identi¢ed line) is a very signi¢cant similar change to in theconformation of the bac- 2 phate analogue. role has been establishedsignal for elicited the protein in from the dUDP-nativeE. coli and terial dUTPase enzyme induced complex exclusively (Fig. in 2B, the presence of Mg by the lentivirus of equine infectious anemia: loss of the motif the K,L-imido-triphosphate analogue of dUTP but not by The di¡erence signals proved to be saturable (Fig. 3)or allow- replacement of itsdashed conserved line, residues positive is deleterious peak for at 270dUDP. nm), This while conformational the K,L change,-imido- most probably, re£ects ing determination of apparent dissociation constants, Kd;app dUTP-native dUTPase complex is characterized by a negative (Table 1). The result that the triphosphate analogue binds ellipticity peak at 260 nm (Fig. 2B, solid line). The conclusion signi¢cantly (about 10-fold) more strongly to the enzyme is that loss of the C-terminus precludes formation of the bind- than dUDP demonstrates the importance of the Q-phosphate ing pattern characteristic for K,L-imido-dUTP and the tri- moiety of the analogue in the interaction with the enzyme. phosphate analogue-induced protein conformational change. Both far and near UV CD gave, within experimental error, The strength of complex formation between K,L-imido-dUTP the same Kd;app for the K,L-imido-dUTP-dUTPase complex, and truncated dUTPase is in accordance with this conclusion: corroborating that the CD signals describe the interaction the apparent dissociation constant is 10 WM, close to that of adequately. No di¡erence spectra could be recorded if either the dUDP-dUTPase complex (Table 1). 2 Mg or the analogues were omitted from the mixtures (data Trypsinolysis of E. coli dUTPase leads to a rapid decrease 2 not shown), consistent with an interaction of the Mg com- in enzyme activity [13]. The presence of the triphosphate ana- plexes of the analogues with the enzyme. logue K,L-imido-dUTP during trypsinolysis has a signi¢cant protective e¡ect (Fig. 4). The apparent ¢rst order rate con- 3.2. Involvement of the C-terminus in the binding of stant for the activity loss, probably re£ecting the speci¢c hy- K,L-imido-dUTP drolysis of the Arg-Gly peptide bond (residues 141^142), was Speci¢c tryptic hydrolysis at Arg-141 in E. coli dUTPaseFig. 4. K,L-Imido-dUTPestimated protects against to tryptic 0.038 digestion min at31 Arg-141.and 0.0082 Native dUTPase min3 (1201,W inM) the was digested absence in the or absence (closed symbols) or presence of ligand (200 WM, open symbols). Aliquots were taken from the reaction mixture at time intervals and assayed for enzyme activity. removes the C-terminal 11 residues, comprising most ofData Motif were ¢tted to ¢rstpresence order reactions of (solidK,L lines).-imido-dUTP, respectively. The Arg-Gly pep- 5 [13]. The truncated protein does not show any conforma- tide bond is part of the £exible C-terminus and the protective tional change upon complexation with K,L-imido-dUTP as e¡ect exerted by the triphosphate analogue reinforces the con- monitored by far UV CD (Fig. 1B, dotted line). Binding of clusion that the C-terminus is involved in binding of K,L-imi- the analogue can still be followed by near UV CD. However, do-dUTP. FEBS 19693 8-1-98 Table 1 Interaction of di- and triphosphate substrate analogues with dUTPase dUTPase Ligand Technique Kd;app (WM) v[3] (mdeg/WM/cm) Native K,L-imido-dUTP near UV CD (260 nm) 1 þ 0.4 0.06 þ 0.01 far UV CD (206 nm) 1.1 þ 0.5 4.2 þ 1.5 kinetics [14] 5 ^ dUDP near UV CD (270 nm) [12,13] 10^15 0.07^0.09 kinetics [15] 15 ^ Truncated K,L-imido-dUTP near UV CD (270 nm) 10 þ 3 0.08 þ 0.02 dUDP near UV CD (270 nm) [12,13] 15 0.07

FEBS 19693 8-1-98 Summary of CD label-free probe of secondary structure rich insight into folding mechanisms

Caveats low resolution can be tricky to assign changes, esp. near-UV Outline

kNR

N k intensity/quantum yield Φ = emitted = R quenching, FRET Nabsorbed kR + kNR spectral shifts excited-state lifetime anisotropy/polarization Fluorimeter Schematic

chemwiki.ucdavis.edu Excitation & Emission Spectra

Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Ed. 1846 C.A. Royer et al.

fluorescence changes in thewild-type protein to theindi- vidual tryptophan residues. In turn, the results lead to 1500 conclusions about structural changes responsible for fluorescence perturbations.

Results p 750 Steady-state fluorescence unfolding profiles C a, -C IntrinsicThe steady-state fluorescenceTrp Fluorescence: emission spectra of W99F Folding and W19F were measured as a function of urea concen- 375 tration between 0 and 9 M urea (Fig.2). The emission of Trp 19 in W99Fat 0 M urea is, as determined previously 0 (Royer, 1992), quite blue shifted, with a maximum near 3 10 3 45 380 415 45 0 319 nm (Fig. 2A). The spectrum shifts significantly to lon- Wavelength (nrn) ger wavelengths, and there is an overall decrease in the 1846 C.A. Royer et al. 362 fluorescence intensity as urea concentration increases -El (Fig. 2A). The effect of urea on Trp99 in W19F is quite fluorescence changes in thewild-type protein to theindi- different (Fig. 2B). The spectrum in the native state is red- B vidual tryptophan residues. In turn, the results lead to der1500 than that of Trp 19, and the quantum yield is much 900 r conclusions about structural changes responsible for flu- lower (Royer, 1992). In contrast to W99F, a large increase orescence perturbations. in intensity and a significant red shift is observed when 675 W19F is unfolded by urea. The red shift in emission for h =! Results these proteins was determined by calculating the intensity- a averaged emission wavelength, (A), using the equation p 750 Steady-state fluorescence unfolding profiles C -a, C The steady-state fluorescence emission spectra of W99F - and W19F were measured as a function of urea concen- 375 2 25 tration between 0 and 9 M urea (Fig.2). The emission of Trp 19 in W99Fat 0 M urea is, as determined previously This quantity is less prone to instrumental noise than is (Royer, 1992), quite blue shifted, with a maximum near 0 0 the peak3 10 maximum3 45 because380 it is an integral415 measurement.45 0 3 IO 3 45 380 415 450 319 nm (Fig. 2A). The spectrum shifts significantly to lon- Wavelength (nrn) It is also a more sensitive value because it arises from a Wavelength (nm) ger wavelengths, and there is an overall decrease in the calculation involving the entire spectrum, and thusit re- fluorescence intensity as urea concentration increases - 362 Fig. 2. Evolution of the intrinsic tryptophan emission spectrum as a flects changes in the shape-El of the spectrum as well as in function of urea concentration between 0 and 9 M urea. Insets repre- (Fig. 2A). The effect of urea on Trp99 in W19F is quite Peakposition. position, The smaller intensity red shift in and the average shape emission can all sentreﬂect the average environment emission wavelength aroundcalculated from ﬂuorophore the spectra and different (Fig. 2B). The spectrum in the native state is red- B wavelength for W19F, 12 nm, versus the 22-nmshift fit to a two-state denaturation transition.A: W99F mutant, 5.5 pM in der than that of Trp 19, and the quantum yield is much 900 r dimer; spectra are 0-9 M urea in 1 M urea steps alternating solid and found for W99Freflects the bluer emission of Trp 19 in lower (Royer, 1992). In contrast to W99F, a large increase dashed lines; numbered spectra correspond to (1) 0 M urea, (2) 3 M urea, the native state. At 9 M urea, the steady-state emission in intensity and a significant red shift is observed when (3) 4 M urea, (4) 5 M urea, and (5) 9 M urea. Between 5 and 9 M urea, spectra675 of the two tryptophanresidues are nearly identi- the intensitiesRoyer increase. et Theal. totalProtein intensities Sci.corresponding 2:1844 to the peak(1993) W19F is unfolded by urea. The red shift in emission for h integrals of these spectra are plotted in Figure 3A. B: W19F mutant, these proteins was determined by calculating the intensity- =!cal, as would be expected for fully solvated residues, a Preliminary fit of fluorescence data from the single 6.1 +M in dimer; spectra are 0-9 M urea in 1 M urea steps alternating averaged emission wavelength, (A), using the equation solid and dashed lines; numbered spectra correspond to (1) 0 M urea, tryptophan mutants to a two-state model (Fig. 2, insets) (2) 4 M urea, (3) 5 M urea, and (4) 9 M urea. Between 5 and 9 M urea, (Gittelman & Matthews, 1990; Fernando & Royer, 1992b) the intensities increase. The total intensities corresponding to the peak yield apparent free energies of folding in theabsence of integrals of these spectra are plotted in Figure 4A. Excitation was at denaturant2 25 of 18.4 t 1.1 and 20.7 * 0.8 kcal/mol (dimer), 295 nm. respectively, for W19F andW99F. The averageemission wavelength data were fit by a model in which quantum This quantity is less prone to instrumental noise than is 0 the peak maximum because it is an integral measurement. yield 3differences IO 3 between45 the380 native and415 denatured450 states It is also a more sensitive value because it arises from a were taken into account.Wavelength These (nm)values are in the same ties measured at320, 350, and 380 nm, respectively. The range as that obtained for the wild-type protein, 18.2 intensity profile observed depends upon the wavelength calculation involving the entire spectrum, and thusit re- Fig. 2. Evolution of the intrinsic tryptophan emission spectrum as a flects changes in the shape of the spectrum as well as in functionkcal/mol, of urea using concentration the same between method 0 and(see 9 Fig. M urea. 1 in Insets the accom- repre- at which the observation is made because the emission position. The smaller red shift in the average emission sentpanying the average paper emission by Mann wavelength et al. calculated [ 19931). from the spectra and wavelength shifts with unfolding. The intensity at 320 nm wavelength for W19F, 12 nm, versus the 22-nmshift fit toThe a two-state evolution denaturation of the total transition. intensityA: W99F of the mutant, 5.5emission pM in decreases in a sigmoidal fashion between 2 and 6 M urea; dimer; spectra are 0-9 M urea in 1 M urea steps alternating solid and found for W99Freflects the bluer emission of Trp 19 in spectrum of Trp 19, i.e., in W99F asa function of urea, the native and unfolded baselines are almost independent dashedis shown lines; numbered in Figure spectra 3A; correspond Figure3B-D to (1) displays 0 M urea, (2)the 3 Mintensi- urea, of denaturant concentration. Although the intensity at the native state. At 9 M urea, the steady-state emission (3) 4 M urea, (4) 5 M urea, and (5) 9 M urea. Between 5 and 9 M urea, spectra of the two tryptophanresidues are nearly identi- the intensities increase. The total intensities corresponding to the peak cal, as would be expected for fully solvated residues, integrals of these spectra are plotted in Figure 3A. B: W19F mutant, Preliminary fit of fluorescence data from the single 6.1 +M in dimer; spectra are 0-9 M urea in 1 M urea steps alternating solid and dashed lines; numbered spectra correspond to (1) 0 M urea, tryptophan mutants to a two-state model (Fig. 2, insets) (2) 4 M urea, (3) 5 M urea, and (4) 9 M urea. Between 5 and 9 M urea, (Gittelman & Matthews, 1990; Fernando & Royer, 1992b) the intensities increase. The total intensities corresponding to the peak yield apparent free energies of folding in theabsence of integrals of these spectra are plotted in Figure 4A. Excitation was at denaturant of 18.4 t 1.1 and 20.7 * 0.8 kcal/mol (dimer), 295 nm. respectively, for W19F andW99F. The averageemission wavelength data were fit by a model in which quantum yield differences between the native and denatured states were taken into account. These values are in the same ties measured at320, 350, and 380 nm, respectively. The range as that obtained for the wild-type protein, 18.2 intensity profile observed depends upon the wavelength kcal/mol, using the same method(see Fig. 1 in the accom- at which the observation is made because the emission panying paper by Mann et al. [ 19931). wavelength shifts with unfolding. The intensity at 320 nm The evolution of the total intensity of the emission decreases in a sigmoidal fashion between 2 and 6 M urea; spectrum of Trp 19, i.e., in W99F asa function of urea, the native and unfolded baselines are almost independent is shown in Figure 3A; Figure3B-D displays the intensi- of denaturant concentration. Although the intensity at Excited State Lifetimes by TCSPC (Time-Correlated Single Photon Counting)

time (ns) Wahl, Technical Note: TCSPC v2.1, Picoquant TCSPC: Mixed Systems

monoexponential biexponential

⎡ t ⎤ n(t) = n0 exp − ⎣⎢ τF ⎦⎥

t = τF for a single decay event Frequency-Domain Lifetime Measurements Fluorescence Quenching

dynamic quenching – shorter lifetime

static quenching – no effect on lifetime Environment-Sensitive Extrinsic Fluors: the Twisted Intramolecular Charge-Transfer Mechanism

rotation (conformation relaxation)

conﬁned environment or high viscosity slows rotation, raises quantum yield

examples: ANS, TNS, Nile Red, Thioﬂavin T… Fluorescence Quenching: Stern-Volmer Plots

dynamic static mixed

F0/F

�0/�

F τ dynamic 0 0 -- kinetics! = =1+ KSV [Q] =1+ kaτ 0 [Q] F τ F Q static 0 [ ] -- afﬁnity! =1+ KSV [Q] =1+ F KD Förster Resonance Energy Transfer: A Special Case of Dynamic Quenching

non-radiative (dipole-dipole coupling) 120 donor acceptor

100

20 Normalized Absorbance/Fluorescence Normalized 0 400 450 500 550 600 650 700 Wavelength (nm)

J decrease in donor lifetime overlap in donor emission and acceptor excitation Förster Resonance Energy Transfer: A Special Case of Dynamic Quenching

Förster equation describes distance dependence of energy transfer

efﬁciency, ETeff: 120 donor acceptor 100 A 1 80 ETeff = = 6 A + D 1+ r R 60 ( 0 ) 2 −4 40 R0 ∝κ ΦD Jn

20 Normalized Absorbance/Fluorescence Normalized 0 Förster radius R depends on: 400 450 500 550 600 650 700 0 2 Wavelength (nm) orientational parameter � ≈ 2/3

donor quantum yield �D J overlap integral J overlap in donor emission refractive index n and acceptor excitation Nath et al. Supporting Material: Conformational ensembles of S and tau

Distance Measurement by FRET 1 ETeff = 6 very sensitive to distances ~ Nath et al. Supporting Material: Conformational1 ensembles+(r R0 of) S and Rtau0 (typically 20-60Å)

Figure S2. Mean Rg values obtainedrapid bydynamics ECMC of can tau ﬂatten in solution using 0, 6 or 12 smFRET constraints, demonstrating similarthe asymptoticdistance dependence behavior to S (cf. Figure 2a): most of the compaction relative to the random-coil limit is observed even with a partial set of 6 constraints.

�2 = 2/3? not always!

Figure S3. (Top) Distribution of values for the orientation factor κ2 based on 1000 samples of

random values for the three angles describing dye geometry θD, θD and ϕ on the interval [0, 2π], and typical experimental anisotropy values for labeled protein. (Bottom) Distribution of 2 perturbations in Förster radius R0 due to the potential uncertainty in κ . Figure S1. (a) Inter-residue distance distribution P(r) of an unconstrained excluded-volume MC simulation as a function of sequence separation. Mean and standard deviation of P(r) are displayed as black and gray lines, respectively. Inset: plot of P(r) distribution at a sequence separation of 100 residues (red), demonstrating that the distributions from excluded-volume MC calculations are much better described by normal distributions (black) than by the Gaussian chain model (orange). (b) Relationship between inter-dye distance and ETeff, calculated using the Förster equation alone (black), the excluded-volume approach described here (red), and the Gaussian chain model (blue). (c) The expected means and standard deviations of P(r) distributions corresponding to different values of ETeff. These mean and standard deviation values were used to parametrize harmonic constraints for ECMC calculations based on measured ETeff values.

Page 7 of 8

Page 6 of 8 238 Subtle Conformational Biases in α-Synuclein Molecules

Temperature dependence of the intramolecular diffusion coefficients

The probes' fluorescence decay curves obtained in trFRET experiments are affected by nanosecond fluctuations of the distance between the probes. Analysis of the experimental decay curves assumed Distance Distributions by TR-FRET a model of normal diffusion of the labeled residues with constraints imposed by the segmental EED distributions. We interpreted the diffusion coefficients determined by analysis of the trFRET exper- 238 Subtle Conformational Biases in α-Synuclein Moleculesiments as a measure of the fast fluctuations of the distance between the labeled residues. These fluctuations result from the constrained fast Brownian Temperature dependence of the intramolecularmotions of residues that compose the labeled D diffusion coefficients segment, regardless of the rotational or translational DA motions of the entire chain. At elevated tempera- The probes' fluorescence decay curves obtainedtures, in the rate of fluctuation is expected to be trFRET experiments are affected by nanosecondincreased for all chain segments, while the con- fluctuations of the distance between the probes.straints that are dependent on specific interactions Analysis of the experimental decay curves assumedmay vary to different extents in each segment. For a model of normal diffusion of the labeled residuesmost segments, the expected monotonous linear with constraints imposed by the segmentalincrease EED of the diffusion coefficient with tempera- distributions. We interpreted the diffusionture coeffi- was observed up to very high values of close to cients determined by analysis of the trFRET25Å exper-2/ns (i.e., 2.5×10− 6 cm2/s). These are very high iments as a measure of the fast fluctuationsintramolecular of the diffusion coefficient values, only an distance between the labeled residues. Theseorder fluc- of magnitude smaller than the common free tuations result from the constrained fast Browniandiffusion coefficients forlow-molecular-weight Fig. 3. Globalmotions analysis of of residues trFRET decay that curves compose to yield the labeledcomponents in aqueous solution. This high value intramolecularsegment, distance regardless distributions. of the (a) rotational Fluorescence or translationalwas independent of segment length (beyond some decay curvesmotions of the segmentof the entire labeled chain. at positions At elevated 4 and tempera-threshold length) and appears to set an upper limit 18, at 5 °C (blue)tures, and the 40 rate °C (red). of The fluctuation upper curve is at expected each for to the be segments of αS. This pattern of temperature temperatureincreased was measured for all for chain the DO segments, mutant, and while the thedependence con- is as expected for the unconstrained lowerGrupi curvestraints for& Haas. the DA that J. mutant. are Mol. dependent TheBiol. major 411:234 on change specific (2011) of the interactions donor fluorescence lifetime induced by the distance- dynamics of the ends of unstructured polypeptide dependentmay excitation vary energy to different transfer extents and the in enhanced each segment.segments. For There were three exceptions to this deviation frommost monoexponential segments, the decay expected is visible. monotonous The normal linear temperature effect. The diffusion coefficient lower frameincrease shows the of autocorrelation the diffusion of coefficient the residuals with for tempera-obtained for the shortest labeled segment, only nine the two DAture experiments. was observed (b) Fitting up to the very decay high data values of the of closeresidues to long (18 to 26), was very low, and no DO and DA25Å mutants2/ns (i.e., to a 2.5 distance×10− 6 cm distribution2/s). These model are verytemperature high dependence could be observed. This using the globalintramolecular analysis approach diffusion yields coefficient the distance values, onlyresult an seems to be significant, even though for this distributionsorder at 5 °C of (blue) magnitude and 40 smaller °C (red). than the commonshort free segment the transfer efficiency was high and diffusion coefficients forlow-molecular-weightthe donor lifetime was short. At the reduced lifetime Fig. 3. Global analysis of trFRET decay curves to yield components in aqueous solution. This highof value the excited state, the relative contribution of the intramolecular distance distributions. (a) Fluorescencesummarizedwas in independentFig. 5, where of segment it is evident length that (beyondfast some fluctuations to the rate of energy transfer and decay curves of the segment labeled at positionswithin 4 and thethreshold experimental length) uncertainty, and appears the to mean set an of upperhence limit to the overall excited state decay rate is 18, at 5 °C (blue) and 40 °C (red). The upper curve ateach each distancefor the distribution segments of wasαS. unaffected This pattern by of the temperaturereduced. Thus, the uncertainty in the determination temperature was measured for the DO mutant, andchange the independence temperature. is Thus, as expected over the temperaturefor the unconstrainedof the value of the diffusion is larger than that found lower curve for the DA mutant. The major changerange of the of 5 to 40 °C, the N-terminal domain of αS for the other segments. The temperature depen- donor fluorescence lifetime induced by the distance- dynamics of the ends of unstructured polypeptide dependent excitation energy transfer and the enhancedneither foldssegments. nor unfolds. There Only were for three two segments exceptions todence this of the diffusion coefficient obtained for the deviation from monoexponential decay is visible.was The an increasenormal temperaturein the width effect. of the The distribution diffusion coefficientterminal chain section, 4–18, is nonlinear. It seems lower frame shows the autocorrelation of the residualsobserved. for obtained Thus, except for the for shortest the segment labeled labeled segment, in onlythat nine this chain section undergoes a minor unfolding the two DA experiments. (b) Fitting the decay datathe of the NAC domainresidues (66 long–90) (18 and to the 26), N-terminal was very (4–18) low, andtransition no between 20 and 30 °C that is manifested DO and DA mutants to a distance distributionchain model segments,temperature the dependence chain segments could be of observed. the by This a very small increase in the width of the distance using the global analysis approach yields the distanceN-terminalresult domain seems are to be fully significant, disordered even under though fordistribution this and relaxation of the forces that distributions at 5 °C (blue) and 40 °C (red). folding conditions,short segment and the the transfer changes efficiency reported was for highconstrain and the dynamics of the chain ends. At 30 °C, the entirethe molecule donor lifetime (change was in short. the At far-UV the reduced CD lifetimethe diffusion coefficient of the ends of this segment is 35 signal andof the enhanced excited state, rates the of relative aggregation) contributionthe of the same as for the remaining segments; that is, summarized in Fig. 5, where it is evidentcannot that befast attributed fluctuations to to major the rate changes of energy of the transferthe and specific local constraints were overcome by the within the experimental uncertainty, the meandisordered of hence state to of thisthe domain.overall excited state decay rateincreased is temperature. The third exception to the each distance distribution was unaffected by the reduced. Thus, the uncertainty in the determination change in temperature. Thus, over the temperature of the value of the diffusion is larger than that found range of 5 to 40 °C, the N-terminal domain of αS for the other segments. The temperature depen- neither folds nor unfolds. Only for two segments dence of the diffusion coefficient obtained for the was an increase in the width of the distribution terminal chain section, 4–18, is nonlinear. It seems observed. Thus, except for the segment labeled in that this chain section undergoes a minor unfolding the NAC domain (66–90) and the N-terminal (4–18) transition between 20 and 30 °C that is manifested chain segments, the chain segments of the by a very small increase in the width of the distance N-terminal domain are fully disordered under distribution and relaxation of the forces that folding conditions, and the changes reported for constrain the dynamics of the chain ends. At 30 °C, the entire molecule (change in the far-UV CD the diffusion coefficient of the ends of this segment is signal and enhanced rates of aggregation)35 the same as for the remaining segments; that is, cannot be attributed to major changes of the the specific local constraints were overcome by the disordered state of this domain. increased temperature. The third exception to the Fluorescence Anisotropy z sample excited with linearly polarized light – excitation emission will also be polarized. polarizer* how polarized? depends on how quickly the light x ﬂuorophore is tumbling. source I − I # I − I & r = || ⊥ p = || ⊥ I┴ % ( I|| + 2I⊥ $ I|| + I⊥ ' װy I anisotropy polarization selectable emission polarizer*

excitation light Helicase Activity Monitored by Anisotropy

anisotropy jumps upon helicase binding to labeled DNA

ATP addition triggers unwinding – anisotropy progressively decreases as strands unwind

low final anisotropy reflects lower molecular weight and higher flexibility of ssDNA product vs. dsDNA substrate

Xu et al. Nuc. Acids Res. e70 (2003) The Perrin Equation

r0 τ r0 τ = ﬂuorescence lifetime =1+ ⇒ r = θ = rotational correlation time r θ τ r = fundamental (maximum) anisotropy 1+ 0 θ

3cos2 ξ −1 � = angle between excitation and emission r = ≤ 0.4 0 5 dipole moments

R = gas constant T = temperature ηVH −1 η = viscosity θ = = (6DR ) RT VH = volume of molecule DR = rotational diffusion coefﬁcient Time-Resolved Anisotropy View Article Online

100000 PCCP Paper

VV(t) 10000 of the slow motion of Lz-A488 to the whole rotation of VH(t) the protein. The segmental correlation time (fseg = 68 ps) 1000 reflects an average of the fast localized motions of the covalently

bound fluorescent dye. Since these are restricted, and con- Intensity [Counts] IRF 100 sequently the randomization of orientations is not achieved, the anisotropy at long times should not decay to zero. However, 10 the overall rotation of the protein is responsible for the decay

1 of its fluorescence anisotropy to zero. The range of angular 024681012141618202224 displacement of these motions can be derived from S assuming Time [ns] seg a‘‘wobbling-in-cone’’model,38 in which the transition dipole Kapusta et al. J. Fluorescence (2003) 13:179 moment of the dye is assumed to move freely within a cone with

afixedhalf-angle.Inthisframework,thehalf-angle,yseg,ofthe cone within which the segment containing the covalently attached dye freely rotates is given by

cos y = 1[(8S + 1)1/2 1] (23) seg 2 seg À

An angle of yseg = 201 was obtained for Lz-A88 in buﬀer, implying that this segment experiences relatively small angular Fig. 3 Fluorescence anisotropy decays from free and membrane-bound Melo et al. Phys. Chem. Chem. Phys. (2014) 16:18105 monomeric Lz-A488. The blue and red solid lines are the best fitting displacements with respect to the protein as a whole during curves of eqn (6) to the anisotropy decays obtained for Lz-A488 in buﬀer the A488 excited state lifetime. The constraints on the local (1.5 mM, DoL = f = 0.21) and in the presence of 4 mM POPC:POPS 80:20 motions of the dye are due to the short covalent linkage LUVs (0.5 mM, DoL = f = 0.21), respectively (see Table 1). The residuals of between the dye and the protein and steric hindrances caused each fit are also shown. by the protein surface,27 as well as to the probable electrostatic interactions between the negatively charged dye and lysozyme.39

Finally, the time-zero anisotropy (r(0) = b1 + b2 = 0.39 (Table 1)) independent depolarizing processes. The fast correlation time was similar to the one measured for A488 in a rigid environment Fig.2: VV and VH polarized decays of Coumarin 6 in ethylene isglycol attributed and the instrument to theresponse rapid restricted movements resulting from 40 function (IRF). The anisotropy decay curve as calculated by FluoFit, assuming G = 1, is plotted at the (ro = 0.376). bottom panel. An appropriate portion of r(t) was fitted to a singlelocal exponential motions decay model by the without covalently linked probe and/or mobility of reconvolution. The fitted curve and the properly weighted residuals are also shown. See the text for To confirm that the main factor responsible for the rather further details. the protein segment to which it is attached, r (t), while the fast high steady-state anisotropy value measured for Lz-A488 in slow correlation time is related to the overall protein rotation in aqueous solution was the limited range of local/segmental solution, r (t):36,37 slow motions presented by the covalently bound A488 probe, lyso- © PicoQuant 2002 TechNote FT-Anisotropy Page 4 r(t)=rslow(t) rfast(t) (18) zyme was also derivatized with BODIPY-FL, a neutral fluoro- Á phore with a slightly longer covalent linker.24 The steady-state where fluorescence anisotropy of Lz-BODIPY in aqueous solution was r (t) = exp( t/ ) (19) lower than the value measured under the same conditions for Published on 26 March 2014. Downloaded by University of Washington 20/01/2016 02:19:52. slow fglobal À Lz-A488 ( r = 0.073 0.070). This result can be partially h i Æ and explained by the longer intensity-weighted mean lifetime of

2 2 Lz-BODIPY as compared to Lz-A488 ( t 2 = 5.5 ns versus 3.4 ns). rfast(t)=r(0)[(1 Sseg ) exp( t/fseg)+Sseg ] (20) h i À Á À The time-resolved anisotropy decay of Lz-BODIPY in aqueous

Here, Sseg is the order parameter characterizing the restricted solution was also described by two correlation times (f1 = 0.33 ns

range of internal angular fluctuations of the protein segment and f2 = 3.8 ns) with similar amplitudes (b1 = 0.14 and b2 = 0.13) 2 containing the covalently bound dye (Sseg = b2/(b1 + b2)). The (Fig. S1, ESI†). In this case, the apparent time-zero anisotropy

short and long rotational correlation times obtained from the fit (r(0) = b1 + b2 = 0.27) was significantly lower than the funda- 41 (Table 1) are, respectively, related to fseg and fglobal by mental anisotropy reported for the free dye (ro = 0.370),

1 indicating that there are ultrafast motions of the covalently- 1 1 À bound dye relative to the native protein which cannot be f1 (21) ¼ fseg þ fglobal! observed within the time resolution of our instrumental setup. In addition, the half-angle associated to the ‘‘wobbling’’ motion

of BODIPY covalently-tagged to the protein was higher (yseg =381) f2 = fglobal (22) than the one obtained for Lz-A488, confirming our previous

The recovered value for fglobal = 5.2 ns is in good agreement reasoning. Note that due to the lower amplitude associated to with the theoretical prediction from the Perrin equation for the the whole rotation of the protein, it was diﬃcult to recover its overall rotation in aqueous solution of a rigid hydrated sphere correlation time, which was lower than the one obtained for

(f = 5.5 ns for the native lysozyme (Mr = 14.3 kDa) in aqueous Lz-A488 or the theoretically expected value. solution at 23 1C, and assuming 20% hydration and a partial We next sought to obtain information about the rota- 3 1 27 specific volume of 0.74 cm gÀ ), confirming the assignment tional properties of the monomeric membrane-bound Lz-A488.

This journal is © the Owner Societies 2014 Phys. Chem. Chem. Phys., 2014, 16, 18105--18117 | 18111 Summary of Fluorescence sensitive, selective and versatile probe of: conformation, local environment, intramolecular distances broad dynamic range for kinetics: excited-state lifetime, anisotropy, stopped-ﬂow

Caveats many factors can affect intrinsic ﬂuorescence extrinsic ﬂuorophores may perturb the system distance determination – low resolution, many potential artifacts