DOCSLIB.ORG

REVIEW Small-Angle Scattering for Structural Biology—Expanding the Frontier While Avoiding the Pitfalls

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
REVIEW Small-Angle Scattering for Structural Biology—Expanding the Frontier While Avoiding the Pitfalls REVIEW Small-angle scattering for structural biology—Expanding the frontier while avoiding the pitfalls David A. Jacques and Jill Trewhella* School of Molecular and Microbial Biosciences, The University of Sydney, Sydney, New South Wales 2006, Australia Received 23 December 2009; Revised 17 January 2010; Accepted 19 January 2010 DOI: 10.1002/pro.351 Published online 21 January 2010 proteinscience.org Abstract: The last decade has seen a dramatic increase in the use of small-angle scattering for the study of biological macromolecules in solution. The drive for more complete structural characterization of proteins and their interactions, coupled with the increasing availability of instrumentation and easy-to-use software for data analysis and interpretation, is expanding the utility of the technique beyond the domain of the biophysicist and into the realm of the protein scientist. However, the absence of publication standards and the ease with which 3D models can be calculated against the inherently 1D scattering data means that an understanding of sample quality, data quality, and modeling assumptions is essential to have confidence in the results. This review is intended to provide a road map through the small-angle scattering experiment, while also providing a set of guidelines for the critical evaluation of scattering data. Examples of current best practice are given that also demonstrate the power of the technique to advance our understanding of protein structure and function. Keywords: Small-angle scattering; neutron scattering; X-ray scattering; protein structure; protein complexes; structural modeling; SAXS; SANS; contrast variation Introduction information has been understood for over 80 years.1 The small-angle scattering of X-rays from macromo- The earliest biological small-angle X-ray scattering lecules in solution and its relationship to basic shape experiments, performed in the 1950s, necessarily used easily purified proteins such as hemoglobin and ovalbumin. Although data interpretation at that Abbreviations BME, b-mercaptoethanol; CD, circular dichroism time was restricted to relatively simple parameters, spectropolarimetry; Dmax, maximum linear dimension; DTT, dithi- othreitol; EM, electron microscopy; HSQC, heteronuclear single such as radius of gyration, it was believed even then quantum coherence; NMR, nuclear magnetic resonance; Rg, ra- that the most important application of small-angle dius of gyration; SDS-PAGE, sodium dodecyl sulfate polyacryl- scattering would be to the study of biological macro- amide gel electrophoresis; TCEP, tris(carboxyethyl) phosphine. molecules.2 In the 1970s and 1980s, the molecular *Correspondence to: Jill Trewhella, Building G08, Butlin Ave, biology revolution and advances in instrumentation University of Sydney, Sydney, NSW 2006, Australia. E-mail: jill. saw some growth in applications of the technique. A [email protected]. significant advance in this period was in the use of Grant sponsor: Australian Research Council (ARC) Discovery small-angle neutron scattering with contrast varia- Project; Grant number: DP09984536; Grant sponsor: Australian Institute of Nuclear Science and Engineering (AINSE) tion to characterize the shapes and dispositions of Postgraduate Research Award. components within bimolecular complexes. Neutron 642 PROTEIN SCIENCE 2010 Published by Wiley-Blackwell. VC 2010 The Protein Society contrast variation revealed that DNA was wrapped scattering experimenter, so that they may avoid around the outside of nucleosome core particles pro- potential pitfalls as the biological applications con- viding an understanding of how DNA was packaged tinue to expand into new territory. in chromosomes3 22 years before the first high-reso- lution crystal structure of the nucleosome appeared.4 The Basics of Small-Angle Scattering Through the 1970s and 1980s, two groups, Engel- It is important for the small-angle scattering experi- man and Moore and coworkers, and May et al., used menter to have a basic understanding of the under- neutron contrast variation to systematically map the lying physics of the technique to avoid being misled. positions of protein and RNA subunits within the We therefore provide a short description of the ribosome leading to a comprehensive map of the 30S essential elements here. subunit5 and a partial map of the larger 50S subu- X-rays and neutrons have properties of plane nit.6 It would take another 8 years before the first waves, that is, amplitude and wavelength, and as high-resolution crystal structure of the large subunit they pass through matter, secondary wavelets are of the ribosome would appear.7 generated by interactions with individual atoms, With the sophistication and, at the time, unique and the resulting coherent scattering can construc- nature of these experiments, the main challenge lay tively or destructively interfere. The diffraction pat- in data interpretation, and experts in theoretical and terns from proteins in crystals that are exploited in computational methods were required for data analy- crystallography are the result of such interference, sis and modeling. These difficulties have largely been between wavelets scattered by atoms within protein overcome by the ready availability of powerful desk molecules that are regularly spaced in the crystal top computers and the development and continual lattice. Small-angle scattering arises from the coher- improvement of data interpretation and modeling ent secondary wavelets that are scattered by atoms tools; today the most widely used is the ATSAS suite within a single molecule, and as a result it is of programs from the Hamburg EMBL (European Mo- observed for molecules in crystals or in solution. lecular Biology Laboratory) group led by Svergun and Although small-angle solution scattering is often coworkers8 as it has been developed explicitly for described as a low-resolution technique (as it does structural biology applications and is readily down- not provide information on atomic coordinates), it is loaded from the web complete with user manuals. more appropriate to describe it as a technique capa- These developments have made small-angle scatter- ble of providing high-precision information with ing accessible to the broader structural biology com- respect to size and shape.9 It is the rotational aver- munity. Consequently, the scope of questions that can aging of the molecules in solution that limits the in- now be answered by small-angle scattering is growing formation content of small-angle scattering more rapidly, and its potential application to biology has than the resolution limits of the experiment. Indeed, never been greater. although crystallographers refer to resolution in As yet, no community-accepted criteria or stand- terms of the highest angle data measured, as it is a ards have been agreed to for the publication of measure of the smallest distances between scatter- small-angle scattering data. In the case of crystallog- ing centers that can be resolved, the small-angle raphy and NMR, the pressure to develop such stand- scattering specialist will refer to the resolution lim- ards grew as the techniques became more widely its more often in terms of the smallest angles for used, and so the development of broadly accepted which data can be measured as it is this resolution standards must also evolve as the use of small-angle limit that determines the longest distances that can scattering continues to grow. As small-angle scatter- be characterized by the data. This effect is a direct ing yields data that are inherently one-dimensional, result of the reciprocal nature of the scattering data and the user invariably is looking to support a with respect to real space dimensions, and the condi- three-dimensional model, overinterpretation of the tion for the minimum angle required to characterize data will always be a risk if not given careful consid- a scattering particle with a given maximum dimen- eration. Importantly, the absence of quality control sion is given below. in sample characterization and data reduction can To interpret scattering data in terms of accurate mislead the experimenter. In this review, we structural parameters, the scattering signal must be describe the small-angle scattering experiment, its measured from a sample of monodisperse, identical range of application, and the necessary quality con- particles. Sample preparation is therefore a critically trol measures. A particular emphasis is given to the important step [Fig. 1(A)] and will be discussed importance of standards for demonstrating sample more completely later. quality, and the power of other biophysical and bio- Although there are several technical challenges chemical methods in providing constraints for mod- associated with the development of small-angle scat- eling that will reduce the likelihood of overinterpre- tering instrumentation, schematically the experi- tation of the scattering data. It is intended that this mental setup is relatively simple [Fig. 1(B)]. A review be a guide to the nonspecialist small-angle highly collimated X-ray or neutron beam is used to Jacques and Trewhella PROTEIN SCIENCE VOL 19:642—657 643 Figure 1. Roadmap through the small-angle scattering experiment. 644 PROTEINSCIENCE.ORG Small-Angle Scattering for Structural Biology illuminate the sample, usually a protein or macro- protein. The P(r) profile is sensitive to the symme- molecular complex in solution (typically >1mg try and domain structure within proteins and as mLÀ1 in 5–30 lL for X-ray
Load more

Recommended publications
  • Developing Back Reflectance Absorbance As a Useful Technique
    Design of a Simple Cryogenic System for Ultraviolet-Visible Absorption Spectroscopy with a Back-reflectance Fiber Optic Probe Andrew Vinyard, Kaj Hansen, Ross Byrd, Douglas A Stuart * and John Hansen * Department of Chemistry, University of West Georgia *Corresponding Authors, email: [email protected] and [email protected] Abstract We report a convenient and inexpensive technique for the rapid acquisition of absorption spectra from small samples at cryogenic temperatures using a home built cryostat with novel collection optics. A cylindrical copper block was constructed with a coaxial bore to hold a 4.00 mm diameter EPR tube and mounted on a copper feed in thermal contact with liquid nitrogen. A 6.35 mm diameter hole was bored into the side of the cylinder so a fiber optic cable bundle could be positioned orthogonally to the EPR tube. The light passing through the sample is reflected off of the opposing surfaces of the EPR tube and surrounding copper, back through the sample. The emergent light is then collected by the fiber optic bundle, and analyzed by a dispersive spectrometer. Absorption spectra for KMnO4 were measured between 400 nm and 700 nm. Absorption intensity at 506 nm, 525 nm, 545 nm and 567 nm was found to be proportional to concentration, displaying Beer’s law like behavior. The EPR tube had an internal diameter of 3.2 mm; the double pass of the probe beam through the sample affords a central path length of about 6.4 mm. Comparing these measurements with those recorded on a conventional tabletop spectrometer using a cuvette with a 10.00 mm path length, we consistently found a ratio between intensities of 0.58 rather than the anticipated 0.64.
    [Show full text]
  • 2 in 1 Fluorescence and Absorbance Spectrometer
    2 in 1 Fluorescence and Application Fluorescence Absorbance Spectrometer: Note How does it work? Life Sciences Figure 1: Duetta (left) and the inside of the Duetta sample compartment (right) with direction of the light path Introduction Duetta™ is a 2-in-1 fluorescence and absorbance excitation energy and the spectrum at longer wavelengths spectrometer from HORIBA Scientific. There are several is acquired to measure the distribution of intensity and benefits of having two different spectroscopies in one energy (wavelength) of the photons emitted. instrument. For high concentration solutions of interest, both primary and secondary inner-filter effects will affect Fluorescence Excitation Spectrum the fluorescence spectrum measured on a standard Acquiring the intensity of photons emitted at a single fluorometer. Having absorbance and fluorescence emission wavelength and scanning the excitation spectroscopy on the same instrument enables Duetta monochromator to excite the population of molecules in and EzSpec software to apply corrections for inner-filter the sample with different wavelengths. A fluorescence effect and provide more accurate data, for a wider range of excitation spectrum is analogous to an absorbance sample concentrations. Another benefit of the two in one spectrum, but is specific to a single emitting species/ instrument is that absorbance results and fluorescence wavelength as opposed to collecting all absorbing species results contain less error since the sample does not move in a sample or solution. from one instrument to another to get both measurements. Standard fluorescence methods such as Emission %Transmittance Spectrum Spectrum and Excitation Spectrum are available on Duetta, This is a ratio, in terms of percentage, of the intensity of but EzSpec software gives a user the ability to measure transmitted light through an absorbing sample (I) compared the Absorbance Spectrum as a stand-alone method or to the transmitted light through a blank solvent (I0).
    [Show full text]
  • Optical and Electron Paramagnetic Resonance Characterization of Point Defects in Semiconductors
    Air Force Institute of Technology AFIT Scholar Theses and Dissertations Student Graduate Works 3-1-2019 Optical and Electron Paramagnetic Resonance Characterization of Point Defects in Semiconductors Elizabeth M. Scherrer Follow this and additional works at: https://scholar.afit.edu/etd Part of the Electromagnetics and Photonics Commons Recommended Citation Scherrer, Elizabeth M., "Optical and Electron Paramagnetic Resonance Characterization of Point Defects in Semiconductors" (2019). Theses and Dissertations. 2463. https://scholar.afit.edu/etd/2463 This Dissertation is brought to you for free and open access by the Student Graduate Works at AFIT Scholar. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of AFIT Scholar. For more information, please contact [email protected]. OPTICAL AND ELECTRON PARAMAGNETIC RESONANCE CHARACTERIZATION OF POINT DEFECTS IN SEMICONDUCTORS DISSERTATION Elizabeth M. Scherrer, Captain, USAF AFIT-ENP-DS-19-M-091 DEPARTMENT OF THE AIR FORCE AIR UNIVERSITY AIR FORCE INSTITUTE OF TECHNOLOGY Wright-Patterson Air Force Base, Ohio DISTRIBUTION STATEMENT A APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED. The views expressed in this thesis are those of the author and do not reflect the official policy or position of the United States Air Force, Department of Defense, or the United States Government. This material is declared a work of the U.S. Government and is not subject to copyright protection in the United States. AFIT-ENP-DS-19-M-091 OPTICAL AND ELECTRON PARAMAGNETIC RESONANCE CHARACTERIZATION OF POINT DEFECTS IN SEMICONDUCTORS DISSERTATION Presented to the Faculty Department of Engineering Physics Graduate School of Engineering and Management Air Force Institute of Technology Air University Air Education and Training Command In Partial Fulfillment of the Requirements for the Doctor of Philosophy Degree Elizabeth M.
    [Show full text]
  • Spectrophotometry Light and Spectra
    Page: 1 Spectrophotometry Spectrophotometry and colorimetry are conventional techniques for quantitatively determining substances encountered in biochemistry. All substances in solution absorb light of some wavelength and transmit light of other wavelengths. Absorbance is a characteristic of a substance just like melting point, boiling point, density and solubility. Absorbance can be related to the amount of the substance in solution, thus it can be used to quantitatively determine the amount of substance that is present. Light and Spectra Light or electromagnetic radiation is composed of photons moving in a wave that oscillates along the path of motion. The wavelength of light is defined as the distance between adjacent peaks in the wave and can be further defined by the equation: λ = c / ν where λ is the wavelength, c is the speed of light, and ϖ is the frequency or number of waves passing a certain point per unit time. Photons of different wavelengths have different energies that are given by: E = hc / λ = h ν where h is Planck's constant. Thus, the shorter the wavelength, the greater the energy. Electromagnetic radiation can be divided into various regions according to wavelength: the ultraviolet region has wavelengths 200-400 nm and the visible region has wavelengths of 400-700 nm. There are other regions such as infrared, radio wave, microwave and more, but you will not apply them in this course. In the visible region, lights of different wavelengths have different colors: violet and blue in the low wavelength region and orange and red in the high wavelength region. When a substance in solution appears blue, it means that the substance is absorbing red light and transmitting blue light.
    [Show full text]
  • Electron Paramagnetic Resonance of Radicals and Metal Complexes. 2. International Conference of the Polish EPR Association. Wars
    ! U t S - PL — voZ, PL9700944 Warsaw, 9-13 September 1996 ELECTRON PARAMAGNETIC RESONANCE OF RADICALS AND METAL COMPLEXES 2nd International Conference of the Polish EPR Association INSTITUTE OF NUCLEAR CHEMISTRY AND TECHNOLOGY UNIVERSITY OF WARSAW VGL 2 8 Hi 1 2 ORGANIZING COMMITTEE Institute of Nuclear Chemistry and Technology Prof. Andrzej G. Chmielewski, Ph.D., D.Sc. Assoc. Prof. Hanna B. Ambroz, Ph.D., D.Sc. Assoc. Prof. Jacek Michalik, Ph.D., D.Sc. Dr Zbigniew Zimek University of Warsaw Prof. Zbigniew Kqcki, Ph.D., D.Sc. ADDRESS OF ORGANIZING COMMITTEE Institute of Nuclear Chemistry and Technology, Dorodna 16,03-195 Warsaw, Poland phone: (0-4822) 11 23 47; telex: 813027 ichtj pi; fax: (0-4822) 11 15 32; e-mail: [email protected] .waw.pl Abstracts are published in the form as received from the Authors SPONSORS The organizers would like to thank the following sponsors for their financial support: » State Committee of Scientific Research » Stiftung fur Deutsch-Polnische Zusammenarbeit » National Atomic Energy Agency, Warsaw, Poland » Committee of Chemistry, Polish Academy of Sciences, Warsaw, Poland » Committee of Physics, Polish Academy of Sciences, Poznan, Poland » The British Council, Warsaw, Poland » CIECH S.A. » ELEKTRIM S.A. » Broker Analytische Messtechnik, Div. ESR/MINISPEC, Germany 3 CONTENTS CONFERENCE PROGRAM 9 LECTURES 15 RADICALS IN DNA AS SEEN BY ESR SPECTROSCOPY M.C.R. Symons 17 ELECTRON AND HOLE TRANSFER WITHIN DNA AND ITS HYDRATION LAYER M.D. Sevilla, D. Becker, Y. Razskazovskii 18 MODELS FOR PHOTOSYNTHETIC REACTION CENTER: STEADY STATE AND TIME RESOLVED EPR SPECTROSCOPY H. Kurreck, G. Eiger, M. Fuhs, A Wiehe, J.
    [Show full text]
  • UV-Vis Spectroscopy: a New Approach for Assessing the Color Index of Transformer Insulating Oil
    sensors Article UV-Vis Spectroscopy: A New Approach for Assessing the Color Index of Transformer Insulating Oil Yang Sing Leong 1 ID , Pin Jern Ker 1,*, M. Z. Jamaludin 1, Saifuddin M. Nomanbhay 1, Aiman Ismail 1, Fairuz Abdullah 1, Hui Mun Looe 2 and Chin Kim Lo 2 1 Institute of Power Engineering, College of Engineering, Universiti Tenaga Nasional, Kajang 43000, Selangor, Malaysia; [email protected] (Y.S.L.); [email protected] (M.Z.J.); [email protected] (S.M.N.); [email protected] (A.I.); [email protected] (F.A.) 2 Tenaga Nasional Berhad (TNB) Research Sdn. Bhd., Bandar Baru Bangi, Kajang 43000, Selangor, Malaysia; [email protected] (H.M.L.); [email protected] (C.K.L.) * Correspondence: [email protected] Received: 30 April 2018; Accepted: 15 June 2018; Published: 6 July 2018 Abstract: Monitoring the condition of transformer oil is considered to be one of the preventive maintenance measures and it is very critical in ensuring the safety as well as optimal performance of the equipment. Various oil properties and contents in oil can be monitored such as acidity, furanic compounds and color. The current method is used to determine the color index (CI) of transformer oil produces an error of 0.5 in measurement, has high risk of human handling error, additional expense such as sampling and transportations, and limited samples can be measured per day due to safety and health reasons. Therefore, this work proposes the determination of CI of transformer oil using ultraviolet-to-visible (UV-Vis) spectroscopy.
    [Show full text]
  • Chemically Denatured Structures of Porcine Pepsin Using Small-Angle X-Ray Scattering
    polymers Article Chemically Denatured Structures of Porcine Pepsin using Small-Angle X-ray Scattering Yecheol Rho 1, Jun Ha Kim 2, Byoungseok Min 2 and Kyeong Sik Jin 2,* 1 Chemical Analysis Center, Korea Research Institute of Chemical Technology, 141, Gajeong-ro, Yuseong-gu, Daejeon 34114, Korea; [email protected] 2 Pohang Accelerator Laboratory, Pohang University of Science and Technology, 80 Jigokro-127-beongil, Nam-Gu, Pohang, Kyungbuk 37673, Korea; [email protected] (J.H.K.); [email protected] (B.M.) * Correspondence: [email protected]; Tel.: +82-54-279-1573; Fax: +82-54-279-1599 Received: 6 November 2019; Accepted: 12 December 2019; Published: 14 December 2019 Abstract: Porcine pepsin is a gastric aspartic proteinase that reportedly plays a pivotal role in the digestive process of many vertebrates. We have investigated the three-dimensional (3D) structure and conformational transition of porcine pepsin in solution over a wide range of denaturant urea concentrations (0–10 M) using Raman spectroscopy and small-angle X-ray scattering. Furthermore, 3D GASBOR ab initio structural models, which provide an adequate conformational description of pepsin under varying denatured conditions, were successfully constructed. It was shown that pepsin molecules retain native conformation at 0–5 M urea, undergo partial denaturation at 6 M urea, and display a strongly unfolded conformation at 7–10 M urea. According to the resulting GASBOR solution models, we identified an intermediate pepsin conformation that was dominant during the early stage of denaturation. We believe that the structural evidence presented here provides useful insights into the relationship between enzymatic activity and conformation of porcine pepsin at different states of denaturation.
    [Show full text]
  • UV-VIS Nomenclature and Units
    IN804 Info Note 804: UV-VIS Nomenclature and Units ​ Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV/VIS) involves the spectroscopy of photons in the UV-visible region. It uses light in the visible and adjacent ​ near ultraviolet (UV) and near infrared (NIR) ranges. In this region of the electromagnetic ​ spectrum, molecules undergo electronic transitions. This technique is complementary to ​ ​ ​ fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state. UV/VIS is based on absorbance. In spectroscopy, the absorbance A is defined as: ​ ​ ​ ​ (1) where I is the intensity of light at a specified wavelength λ that has passed through a sample ​ ​ (transmitted light intensity) and I is the intensity of the light before it enters the sample or ​ ​0 incident light intensity. Absorbance measurements are often carried out in analytical chemistry, ​ ​ ​ ​ since the absorbance of a sample is proportional to the thickness of the sample and the concentration of the absorbing species in the sample, in contrast to the transmittance I / I of a ​ ​ ​ ​0 s ample, which varies exponentially with thickness and concentration. The Beer-Lambert law is ​ used for concentration determination. The term absorption refers to the physical process of absorbing light, while absorbance ​ refers to the mathematical quantity. Also, absorbance does not always measure absorption: if a given sample is, for example, a dispersion, part of the incident light will in fact be scattered by the dispersed particles, and not really absorbed. Absorbance only contemplates the ratio of transmitted light over incident light, not the mechanism by which light intensity decreases.
    [Show full text]
  • Quantifying Protein Concentration Using UV Absorbance Measured by the Ocean HDX Spectrometer by Yvette Mattley, Ph.D
    Quantifying Protein Concentration using UV Absorbance Measured by the Ocean HDX Spectrometer by Yvette Mattley, Ph.D. Application Note KEYWORDS Proteins are a common sample in clinical, diagnostic and re- • Protein search labs. Studies involving protein extraction, purification or • Bovine serum albumin labeling, or working with proteins extracted from cells or labeled for study of the interactions between biomolecules, are com- • Aromatic amino acids mon. Determination of protein concentration is a critical part of protein studies. In this application note, we use an Ocean HDX TECHNIQUES spectrometer to generate a standard curve for bovine serum • UV absorbance albumin (BSA). High sensitivity in the UV, ultra-low stray light performance and upgraded optics make the Ocean HDX ideal • Standard curve for UV absorbance measurements. generation Proteins are central to life. These intricate biomolecules play APPLICATIONS critical roles in cellular processes and metabolism, in catalyz- ing biochemical reactions and serving as structural elements, • Protein concentration and so much more. As vital elements for all life, proteins are determination often the subject of biomedical diagnostic studies and life sci- • Life sciences research ence research. Most research involving proteins, regardless of the answer being sought or the source of the protein, begins Unlocking the Unknown with Applied Spectral Knowledge. with quantifying the amount of protein present in • A(λ) is the absorption of the solution as a func- a sample. tion of wavelength •
    [Show full text]
  • Beer-Lambert Law: Measuring Percent Transmittance of Solutions at Different Concentrations (Teacher’S Guide)
    TM DataHub Beer-Lambert Law: Measuring Percent Transmittance of Solutions at Different Concentrations (Teacher’s Guide) © 2012 WARD’S Science. v.11/12 For technical assistance, All Rights Reserved call WARD’S at 1-800-962-2660 OVERVIEW Students will study the relationship between transmittance, absorbance, and concentration of one type of solution using the Beer-Lambert law. They will determine the concentration of an “unknown” sample using mathematical tools for graphical analysis. MATERIALS NEEDED Ward’s DataHub USB connector cable* Cuvette for the colorimeter Distilled water 6 - 250 mL beakers Instant coffee Paper towel Wash bottle Stir Rod Balance * – The USB connector cable is not needed if you are using a Bluetooth enabled device. NUMBER OF USES This demonstration can be performed repeatedly. © 2012 WARD’S Science. v.11/12 1 For technical assistance, All Rights Reserved Teacher’s Guide – Beer-Lambert Law call WARD’S at 1-800-962-2660 FRAMEWORK FOR K-12 SCIENCE EDUCATION © 2012 * The Dimension I practices listed below are called out as bold words throughout the activity. Asking questions (for science) and defining Use mathematics and computational thinking problems (for engineering) Constructing explanations (for science) and designing Developing and using models solutions (for engineering) Practices Planning and carrying out investigations Engaging in argument from evidence Dimension 1 Analyzing and interpreting data Obtaining, evaluating, and communicating information Science and Engineering Science and Engineering Patterns
    [Show full text]
  • The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly
    crystals Article The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly Alaa Adawy † and Matthew R. Groves * Department of Drug Design, Structural Biology Unit, Groningen Research Institute of Pharmacy, University of Groningen, 9713AJ Groningen, The Netherlands; [email protected] * Correspondence: [email protected]; Tel.: +31-50-36-33305 † Current address: HRTEM laboratory, Scientific and Technical Services, University of Oviedo-CINN, 33006 Oviedo, Spain. Academic Editor: Albert Guskov Received: 6 September 2017; Accepted: 23 October 2017; Published: 31 October 2017 Abstract: High resolution size exclusion chromatography (SEC) coupled with static light scattering (SLS) analyses were conducted to study the effect of the mobile phase ionic strength and protein concentration on the output of SEC experiments. The results highlight the effect of small changes in the mobile phase composition on the estimation of molar masses estimated from retention time-based calibration curve compared with those obtained from SLS analysis. By comparing the SLS data with the SEC chromatograms, we show that SEC can provide helpful information on the protein aggregation state as macromolecules approach known precipitation points in their phase diagrams. This suggests the potential use of SEC as an easily accessible lab-based scanning methodology to monitor protein self-assembly prior to nucleation and crystallization. Implications for the use of SEC to study protein phase diagrams are discussed. Keywords: size exclusion chromatography; static
    [Show full text]
  • Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)
    Journal of Visualized Experiments www.jove.com Video Article Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS) Daniel Some1, Hadar Amartely2, Ayala Tsadok3, Mario Lebendiker2 1 Wyatt Technology Corporation 2 Wolfson Centre for Applied Structural Biology, The Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem 3 Danyel Biotech Ltd. Correspondence to: Daniel Some at [email protected] URL: https://www.jove.com/video/59615 DOI: doi:10.3791/59615 Keywords: Biochemistry, Issue 148, size-exclusion chromatography, multi-angle light scattering (MALS), protein characterization, molecular weight, bovine serum albumin, oligomers, aggregates, protein-protein complexes, quality control Date Published: 6/20/2019 Citation: Some, D., Amartely, H., Tsadok, A., Lebendiker, M. Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS). J. Vis. Exp. (148), e59615, doi:10.3791/59615 (2019). Abstract Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes.
    [Show full text]