Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation
互联网
2240
- Abstract
- Table of Contents
- Figures
- Literature Cited
Abstract
Modern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in sedimentation velocity (SV) size?distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self? and hetero?association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and sedimentation equilibrium (SE), in conjunction with the effective?particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent?solubilized membrane proteins and their interactions by SV and SE. Curr. Protoc. Protein Sci. 71:20.12.1?20.12.49. © 2013 by John Wiley & Sons, Inc.
Keywords: sedimentation equilibrium; sedimentation velocity; chemical equilibria; reversible interaction; size?distribution; multiprotein complex; analytical ultracentrifugation; protein hydrodynamics
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Principles
- Experimental Design and Protocols
- Data Analysis
- Acknowledgements
- Literature Cited
- Figures
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryMaterials
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryFigures
-
Figure 20.12.1 Examples for the shapes of Lamm equation solutions. The profiles are calculated for particles of different size and sedimentation properties. All conditions are calculated for a rotor speed of 50,000 rpm, and 50 concentration profiles are shown at different (in each case equally spaced) time intervals. Later scans are indicated by a higher color temperature. (A ) Small molecules with a sedimentation coefficient of 0.2 S and a diffusion coefficient of 6 × 10−6 cm2 /sec, in Δ t = 300 sec intervals. Similar values are frequently observed for sedimenting buffer salts. (B ) Sedimentation of a peptide of 1 kg/mol and 0.3 S, Δ t = 1000 sec. (C ) A small protein of 10 kg/mol and 1.5 S, Δ t = 500 sec. (D ) A protein of 100 kg/mol and 6 S, Δ t = 300 sec. (E ) Particle of 1 Mg/mol and 30 S, Δ t = 50 sec. (F ) A floating particle with a sedimentation coefficient of –3.0 S and a diffusion coefficient of 2.71 × 10−7 cm2 /sec. Such data patterns may be obtained, for example, with large emulsion or lipid particles. In flotation, the radial dilution is replaced with radial increase in concentration in the plateau region of successive profiles. Figure reproduced from Schuck () View Image -
Figure 20.12.2 Demonstration of interference optical data before (top) and after (bottom) subtracting the estimated TI and RI noise contributions. Both the time‐dependent (radius‐invariant, RI) signal offsets arising from jitter and integral fringe shifts, as well as the radius‐dependent (time‐invariant, TI) signal offset arising from imperfections in the smoothness of the optical elements, can be clearly discerned. After fitting the data with a model including terms for TI and RI noise, the best‐fit values for the TI noise (red line in top panel) and for the RI noise (not shown) can be subtracted from the raw data, as shown in the lower panel. As long as the degrees of freedom for unknown TI and RI noise are maintained in the further analysis, this subtraction does not alter the information content of the data, but allows better visual inspection of the signal from the macromolecular redistribution. View Image -
Figure 20.12.3 Example of SV analyses of a system with non‐interacting species. (A ) Representative subset of the raw data after elimination of systematic noise contributions. (B ) Residuals bitmap from a fit with insufficient quality: the data in (A) are modeled with an impostor single‐species fit, resulting in clearly systematic deviations that can be discerned from the strong diagonal feature in the bitmap. (C ) The quality of the fit with a c ( s ) model results in a residuals bitmap with very few diagonal features. There are some vertical and horizontal lines indicative of the remaining residuals due to technical imperfections in the data acquisition process, such as higher‐order vibrations of optical components. (D ) Size and shape distribution, transformed into coordinates of sedimentation coefficient and molar mass. The color temperature of the contour lines indicates the population of species. Like in one‐dimensional c ( s ), the peak‐width in c ( s , M ) contains contributions both from regularization (reflecting limited resolution given the signal‐to‐noise ratio of the data) and from true heterogeneity. (E ) Reduction of the c ( s , M ) distribution to a pure sedimentation coefficient distribution, general c ( s ~undefined). This is equivalent to a conventional c ( s ) analysis, but without any constraints to a common average frictional ratio of all species. The inset shows a pure molar‐mass distribution, c ( M ~undefined), also derived by integration of c ( s , M ) in a direction orthogonal to c ( s ~undefined). (F ) Size distribution c ( s ) using a hydrodynamic scaling law (black line with broad peaks). Also shown is the result of a Bayesian analysis using prior knowledge in the analysis of this non‐interacting system, here in the form of c ( P δ) ( s ) (blue line with sharp peaks) using the hypothesis that the sample consists of discrete species. Generally, the peak width in c ( s ) can result from either a true polydispersity of the protein (e.g., strong heterogeneity in glycosylation, in conformation, primary sequence, etc.), or from the standard regularization favoring broader peaks for data with low signal/noise ratio. Figure reproduced from Schuck et al. () View Image -
Figure 20.12.4 c(s) distribution of the data shown in Figure . For comparison, the dashed line shows the ls‐undefined(s) distribution derived from a subset of the scans. View Image -
Figure 20.12.5 Example of the multisignal ck (s) analysis of a triple protein mixture of a viral glycoprotein (green), its cognate receptor (blue), and a heterogeneous antigen‐recognition receptor fragment (red). The content of each protein component in the different s ranges is obtained from the global analysis of sedimentation data acquired with the interference optics and with the absorbance system at two different wavelengths (data not shown), using two chromophorically labeled proteins and one unlabeled protein. Solid lines show the c k ( s ) analysis of the triple mixture. The analogous distributions of each protein alone are shown as dashed lines. The formation of two coexisting binary complexes at ∼5 S and ∼7 S and a ternary complex with 1:1:1 stoichiometry at ∼8.5 S can be discerned. Figure reproduced from Schuck et al. () View Image -
Figure 20.12.6 Properties of the reaction boundary A…B as a function of the total loading concentration of A and B, calculated by EPT for the system of Figure . (Top) Velocity of the reaction boundary s A…B following Eq. . (Middle) Composition R A…B of the reaction boundary following Eq.. (Bottom) Fractional signal of the undisturbed boundary, assuming that both components are globular with equal weight‐based extinction coefficients. In all plots the line for the phase transition c Btot View Image
Videos
Literature Cited
| Ansevin, A.T., Roark, D.E., and Yphantis, D.A. 1970. Improved ultracentrifuge cells for high‐speed sedimentation equilibrium studies with interference optics. Anal. Biochem. 34:237‐261. | |
| Aragon, S.R. 2011. Recent advances in macromolecular hydrodynamic modeling. Methods 54:101‐114. | |
| Aragon, S. and Hahn, D.K. 2006. Precise boundary element computation of protein transport properties: Diffusion tensors, specific volume, and hydration. Biophys. J. 91:1591‐1603. | |
| Balbo, A., Minor, K.H., Velikovsky, C.A., Mariuzza, R., Peterson, C.B., and Schuck, P. 2005. Studying multi‐protein complexes by multi‐signal sedimentation velocity analytical ultracentrifugation. Proc. Natl. Acad. Sci. U.S.A. 102:81‐86. | |
| Balbo, A., Brown, P.H., Braswell, E.H., and Schuck, P. 2007. Measuring protein‐protein interactions by equilibrium sedimentation. Curr. Protoc. Immunol. 79:18.8.1‐18.8.28. | |
| Balbo, A., Zhao, H., Brown, P.H., and Schuck, P. 2009. Assembly, loading, and alignment of an analytical ultracentrifuge sample cell. J. Vis. Exp. 5:1530. | |
| Barda‐Saad, M., Shirasu, N., Pauker, M.H., Hassan, N., Perl, O., Balbo, A., Yamaguchi, H., Houtman, J.C., Appella, E., Schuck, P., and Samelson, L.E. 2010. Cooperative interactions at the SLP‐76 complex are critical for actin polymerization. EMBO J. 29:2315‐2328. | |
| Berke, I.C. and Modis, Y. 2012. MDA5 cooperatively forms dimers and ATP‐sensitive filaments upon binding double‐stranded RNA. EMBO J. 31:1714‐1726. | |
| Berkowitz, S.A. 2006. Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals. AAPS J. 8:E590‐605. | |
| Besong, T.M.D., Harding, S.E., and Winzor, D.J. 2012. The effective time of centrifugation for the analysis of boundary spreading in sedimentation velocity experiments. Anal. Biochem. 421:755‐758. | |
| Bevington, P.R. and Robinson, D.K. 1992. Data Reduction and Error Analysis for the Physical Sciences. McGraw‐Hill, New York. | |
| Bothwell, M.A., Howlett, G.J., and Schachman, H.K. 1978. A sedimentation equilibrium method for determining molecular weights of proteins with a tabletop high speed air turbine centrifuge. J. Biol. Chem. 253:2073‐2077. | |
| Brautigam, C.A. 2011. Using Lamm‐Equation modeling of sedimentation velocity data to determine the kinetic and thermodynamic properties of macromolecular interactions. Methods 54:4‐15. | |
| Brookes, E., Cao, W., and Demeler, B. 2009. A two‐dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur. Biophys. J. 39:405‐414. | |
| Brown, P.H. and Schuck, P. 2006. Macromolecular size‐and‐shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys. J. 90:4651‐4661. | |
| Brown, P.H. and Schuck, P. 2008. A new adaptive grid‐size algorithm for the simulation of sedimentation velocity profiles in analytical ultracentrifugation. Comput. Phys. Commun. 178:105‐120. | |
| Brown, P.H., Balbo, A., and Schuck, P. 2007. Using prior knowledge in the determination of macromolecular size‐distributions by analytical ultracentrifugation. Biomacromolecules 8:2011‐2024. | |
| Brown, P.H., Balbo, A., and Schuck, P. 2008. Characterizing protein‐protein interactions by sedimentation velocity analytical ultracentrifugation. Curr. Protoc. Immunol. 81:18.15.1‐18.15.39. | |
| Brown, P.H., Balbo, A., and Schuck, P. 2009. On the analysis of sedimentation velocity in the study of protein complexes. Eur. Biophys. J. 38:1079‐1099. | |
| Brown, P.H., Balbo, A., Zhao, H., Ebel, C., and Schuck, P. 2011. Density contrast sedimentation velocity for the determination of protein partial‐specific volumes. PLoS One 6:e26221. | |
| Cao, W. and Demeler, B. 2008. Modeling analytical ultracentrifugation experiments with an adaptive space‐time finite element solution for multicomponent reacting systems. Biophys. J. 95:54‐65. | |
| Carney, R.P., Kim, J.Y., Qian, H., Jin, R., Mehenni, H., Stellacci, F., and Bakr, O.M. 2011. Determination of nanoparticle size distribution together with density or molecular weight by 2D analytical ultracentrifugation. Nat. Commun. 2:335. | |
| Claverie, J.‐M., Dreux, H., and Cohen, R. 1975. Sedimentation of generalized systems of interacting particles. I. Solution of systems of complete Lamm equations. Biopolymers 14:1685‐1700. | |
| Correia, J.J. and Stafford, W.F. 2009. Extracting equilibrium constants from kinetically limited reacting systems. Methods Enzymol. 455:419‐446. | |
| Cox, D.J. 1965. Computer simulation of sedimentation in the ultracentrifuge. II. Concentration‐independent sedimentation. Arch. Biochem. Biophys. 112:259‐266. | |
| Cox, D.J. 1966. Sedimentation of an initially skewed boundary. Science 152:359‐361. | |
| Dam, J. and Schuck, P. 2004. Calculating sedimentation coefficient distributions by direct modeling of sedimentation velocity profiles. Methods Enzymol. 384:185‐212. | |
| Dam, J., Velikovsky, C.A., Mariuzza, R., Urbanke, C., and Schuck, P. 2005. Sedimentation velocity analysis of protein‐protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s). Biophys. J. 89:619‐634. | |
| Darawshe, S., Rivas, G., and Minton, A.P. 1993. Rapid and accurate microfractionation of the contents of small centrifuge tubes: Application in the measurement of molecular weights of proteins via sedimentation equilibrium. Anal. Biochem. 209:130‐135. | |
| Demeler, B., Saber, H., and Hansen, J.C. 1997. Identification and interpretation of complexity in sedimentation velocity boundaries. Biophys. J. 72:397‐407. | |
| Dishon, M., Weiss, G.H., and Yphantis, D.A. 1967. Numerical simulations of the Lamm equation: III. Velocity centrifugation. Biopolymers 5:697‐713. | |
| Dorwart, M.R., Wray, R., Brautigam, C.A., Jiang, Y., and Blount, P. 2010. S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: Implications for detergent‐solubilized membrane proteins.PLoS Biology 8:e100555. | |
| Durchschlag, H. and Zipper, P. 1994. Calculation of the partial specific volume of organic compounds and polymers. Progr. Colloid. Polym. Sci. 94:20‐39. | |
| Durchschlag, H. and Zipper, P. 1997. Calculation of partial specific volumes and other volumetic properties of small molecules and polymers. J. Appl. Cryst. 30:803‐807. | |
| Ebel, C. 2011. Sedimentation velocity to characterize surfactants and solubilized membrane proteins. Methods 54:56‐66. | |
| Edelstein, S.J. and Schachman, H.K. 1967. The simultaneous determination of partial specific volumes and molecular weights with microgram quantities. J. Biol. Chem. 242:306‐311. | |
| Englund, E.A., Wang, D., Fujigaki, H., Sakai, H., Micklitsch, C.M., Ghirlando, R., Martin‐Manso, G., Pendrak, M.L., Roberts, D.D., Durell, S.R., and Appella, D.H. 2012. Programmable multivalent display of receptor ligands using peptide nucleic acid nanoscaffolds. Nat. Commun. 3:614. | |
| Fujita, H. 1975. Foundations of Ultracentrifugal Analysis. John Wiley & Sons, New York. | |
| Gabrielson, J.P. and Arthur, K.K. 2011. Measuring low levels of protein aggregation by sedimentation velocity. Methods 54:83‐91. | |
| Gabrielson, J.P., Brader, M.L., Pekar, A.H., Mathis, K.B., Winter, G., Carpenter, J.F., and Randolph, T.W. 2007. Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size‐exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity. J. Pharm. Sci. 96:268‐279. | |
| Gabrielson, J.P., Arthur, K.K., Kendrick, B.S., Randolph, T.W., and Stoner, M.R. 2009. Common excipients impair detection of protein aggregates during sedimentation velocity analytical ultracentrifugation. J. Pharm. Sci. 98:50‐62. | |
| Garcia de la Torre, J., Huertas, M.L., and Carrasco, B. 2000. Calculation of hydrodynamic properties of globular proteins from their atomic‐level structure. Biophys. J. 78:719‐730. | |
| Ghirlando, R. 2011. The analysis of macromolecular interactions by sedimentation equilibrium. Methods 54:145‐156. | |
| Ghirlando, R. and Felsenfeld, G. 2008. Hydrodynamic studies on defined heterochromatin fragments support a 30‐nm fiber having six nucleosomes per turn. J. Mol. Biol. 376:1417‐1425. | |
| Ghirlando, R., Litt, M.D., Prioleau, M.‐N., Recillas‐Targa, F., and Felsenfeld, G. 2004. Physical properties of a genomic condensed chromatin fragment. J. Mol. Biol. 336:597‐605. | |
| Harding, S.E., Schuck, P., Abdelhameed, A.S., Adams, G., Kök, M.S., and Morris, G.A. 2011. Extended Fujita approach to the molecular weight distribution of polysaccharides and other polymeric systems. Methods 54:136‐144. | |
| Harrington, W.F. and Kegeles, G. 1973. Pressure effects in ultracentrifugation of interacting systems. Methods Enzymol. 27:106‐345. | |
| Hellman, L.M., Zhao, C., Melikishvili, M., Tao, X., Hopper, J.E., Whiteheart, S.W., and Fried, M.G. 2011. Histidine‐tag‐directed chromophores for tracer analyses in the analytical ultracentrifuge. Methods 54:31‐38. | |
| HeteroAnalysis Contents. 2012. http://www.biotech.uconn.edu/auf/ha‐help/hs2024.htm. | |
| Houtman, J.C., Yamaguchi, H., Barda‐Saad, M., Braiman, A., Bowden, B., Appella, E., Schuck, P., and Samelson, L.E. 2006. Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1. Nat. Struct. Mol. Biol. 13:798‐805. | |
| Inagaki, S., Ghirlando, R., and Grisshammer, R. 2012. Biophysical characterization of membrane proteins in nanodiscs. Methods S1046‐2023(12)00301‐5. | |
| Johnson, M.L. 1992. Why, when, and how biochemists should use least squares. Anal. Biochem. 225:215‐225. | |
| Johnson, M.L. and Straume, M. 1994. Comments on the analysis of sedimentation equilibrium experiments. In Modern Analytical Ultracentrifugation (T.M. Schuster and T.M. Laue, eds.) pp. 37‐65. Birkhäuser, Boston. | |
| Kam, Z., Borochov, N., and Eisenberg, H. 1981. Dependence of laser light scattering of DNA on NaCl concentration. Biopolymers 20:2671‐2690. | |
| Kar, S.R., Kinsbury, J.S., Lewis, M.S., Laue, T.M., and Schuck, P. 2000. Analysis of transport experiment using pseudo‐absorbance data. Anal. Biochem. 285:135‐142. | |
| Lamm, O. 1929. Die Differentialgleichung der Ultrazentrifugierung. Ark. Mat. Astr. Fys. 21B:1‐4. | |
| MacRaild, C.A., Hatters, D.M., Lawrence, L.J., and Howlett, G.J. 2003. Sedimentation velocity analysis of flexible macromolecules: Self‐association and tangling of amyloid fibrils. Biophys. J. 84:2562‐2569. | |
| Nury, H., Manon, F., Arnou, B., le Maire, M., Pebay‐Peyroula, E., and Ebel, C. 2008. Mitochondrial bovine ADP/ATP carrier in detergent is predominantly monomeric but also forms multimeric species. Biochemistry 47:12319‐12331. | |
| Padrick, S.B. and Brautigam, C.A. 2011. Evaluating the stoichiometry of macromolecular complexes using multisignal sedimentation velocity. Methods 54:39‐55. | |
| Padrick, S.B., Deka, R.K., Chuang, J.L., Wynn, R.M., Chuang, D.T., Norgard, M.V., Rosen, M.K., and Brautigam, C.A. 2010. Determination of protein complex stoichiometry through multisignal sedimentation velocity experiments. Anal. Biochem. 407:89‐103. | |
| Padrick, S.B., Doolittle, L.K., Brautigam, C.A., King, D.S., and Rosen, M.K. 2011. Arp2/3 complex is bound and activated by two WASP proteins. Proc. Natl. Acad. Sci. U.S.A. 108:E472‐479. | |
| Philo, J.S. 2000. Sedimentation equilibrium analysis of mixed associations using numerical constraints to impose mass or signal conservation. Methods Enzymol. 321:100‐120. | |
| Provencher, S.W. 1982. A constrained regularization method for inverting data represented by linear algebraic or integral equations. Comput. Phys. Commun. 27:213‐227. | |
| Richards, E.G., Teller, D.C., and Schachman, H.K. 1968. Ultracentrifuge studies with Rayleigh interference optics. II. Low‐speed sedimentation equilibrium of homogeneous systems. Biochemistry 7:1054‐1076. | |
| Rivas, G., Fernandez, J.A., and Minton, A.P. 1999. Direct observation of the self‐association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: Theory, experiment, and biological significance. Biochemistry 38:9379‐9388. | |
| Rivas, G. and Minton, A.P. 2011. Beyond the second virial coefficient: Sedimentation equilibrium in highly non‐ideal solutions. Methods 54:167‐174. | |
| Roark, D.E. 1976. Sedimentation equilibrium techniques: Multiple speed analyses and an overspeed procedure. Biophys. Chem. 5:185‐196. | |
| Rowe, A.J. 2010. Analytical ultracentrifugation: Sedimentation equilibrium. In Encyclopedia of Biophysics (G.C.K. Roberts, ed.). Springer, Berlin. | |
| Rowe, A.J. 2011. Ultra‐weak reversible protein‐protein interactions. Methods 54:157‐166. | |
| Salvay, A.G., Santamaria, M., LeMaire, M., and Ebel, C. 2007. Analytical ultracentrifugation sedimentation velocity for the characterization of detergent‐solubilized membrane proteins Ca++‐ATPase and ExbB. J. Biol. Phys. 33:399‐419. | |
| Schachman, H.K. 1959. Ultracentrifugation in Biochemistry. Academic Press, New York. | |
| Schuck, P. 2000. Size distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78:1606‐1619. | |
| Schuck, P. 2003. On the analysis of protein self‐association by sedimentation velocity analytical ultracentrifugation.Anal. Biochem. 320:104‐124. | |
| Schuck, P. 2004. A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co‐solutes. Biophys. Chem. 108:187‐200. | |
| Schuck, P. 2009. On computational approaches for size‐and‐shape distributions from sedimentation velocity analytical ultracentrifugation. Eur. Biophys. J. 39:1261‐1275. | |
| Schuck, P. 2010a. Diffusion of the reaction boundary of rapidly interacting macromolecules in sedimentation velocity. Biophys. J. 98:2741‐2751. | |
| Schuck, P. 2010b. Sedimentation patterns of rapidly reversible protein interactions. Biophys. J. 98:2005‐2013. | |
| Schuck, P. 2010c. Some statistical properties of differencing schemes for baseline correction of sedimentation velocity data. Anal. Biochem. 401:280‐287. | |
| Schuck, P. 2012. Sedimentation velocity analytical ultracentrifugation. In Encyclopedia of Biophysics (G.C.K. Roberts, ed.). Springer, Berlin. | |
| Schuck, P. and Demeler, B. 1999. Direct sedimentation analysis of interference optical data in analytical ultracentrifugation. Biophys. J. 76:2288‐2296. | |
| Schuck, P. and Rossmanith, P. 2000. Determination of the sedimentation coefficient distribution by least‐squares boundary modeling. Biopolymers 54:328‐341. | |
| Schuck, P., MacPhee, C.E., and Howlett, G.J. 1998. Determination of sedimentation coefficients for small peptides. Biophys. J. 74:466‐474. | |
| Schuck, P., Taraporewala, Z., McPhie, P., and Patton, J.T. 2000. Rotavirus nonstructural protein NSP2 self‐assembles into octamers that undergo ligand‐induced conformational changes. J. Biol. Chem. 276:9679‐9687. | |
| Schuck, P., Perugini, M.A., Gonzales, N.R., Howlett, G.J., and Schubert, D. 2002. Size‐distribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems. Biophys. J. 82:1096‐1111. | |
| Schuck, P., Balbo, A., Brown, P.H., and Zhao, H. 2010. Analytical Ultracentrifugation. In Encyclopedia of Analytical Chemistry (R.A. Meyers, ed.). John Wiley, Chichester. | |
| Schwieters, C.D., Suh, J.‐Y., Grishaev, A., Ghirlando, R., Takayama, Y., and Clore, G.M. 2010. Solution structure of the 128 kDa enzyme I dimer from Escherichia coli and its 146 kDa complex with HPr using residual dipolar couplings and small‐ and wide‐angle X‐ray scattering. J. Am. Chem. Soc. 132:13026‐13045. | |
| Solovyova, A., Schuck, P., Costenaro, L., and Ebel, C. 2001. Non‐ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents. Biophys. J. 81:1868‐1880. | |
| Stafford, W.F. 1992. Boundary analysis in sedimentation transport experiments: A procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal. Biochem. 203:295‐301. | |
| Svedberg, T. and Pedersen, K.O. 1940. The Ultracentrifuge. Oxford University Press, London. | |
| van Holde, K.E. and Baldwin, R.L. 1958. Rapid attainment of sedimentation equilibrium. J. Phys. Chem. 62:734‐749. | |
| Vistica, J., Dam, J., Balbo, A., Yikilmaz, E., Mariuzza, R.A., Rouault, T.A., and Schuck, P. 2004. Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal. Biochem. 326:234‐256. | |
| Yphantis, D.A. 1964. Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297‐317. | |
| Zhao, H., Brown, P.H., Balbo, A., Fernandez Alonso, M.C., Polishchuck, N., Chaudhry, C., Mayer, M.L., Ghirlando, R., and Schuck, P. 2010. Accounting for solvent signal offsets in the analysis of interferometric sedimentation velocity data. Macromol. Biosci. 10:736‐745. | |
| Zhao, H., Balbo, A., Brown, P.H., and Schuck, P. 2011a. The boundary structure in the analysis of reversibly interacting systems by sedimentation velocity. Methods 54:16‐30. | |
| Zhao, H., Brown, P.H., and Schuck, P. 2011b. On the distribution of protein refractive index increments. Biophys. J. 100:2309‐2317. | |
| Zhao, H., Berger, A.J., Brown, P.H., Kumar, J., Balbo, A., May, C.A., Casillas, E., Laue, T.M., Patterson, G.H., Mayer, M.L., and Schuck, P. 2012. Analysis of high‐affinity assembly for AMPA receptor amino‐terminal domains. J. Gen. Physiol. 139:371‐388. | |
| Zhao, H. and Schuck, P. 2012. Global multi‐method analysis of affinities and cooperativity in complex systems of macromolecular interactions. Anal. Chem. 84:9513‐9519. | |
| Zhi, L., Mans, J., Paskow, M.J., Brown, P.H., Schuck, P., Jonjić, S., Natarajan, K., and Margulies, D.H. 2010. Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE‐1 isoforms. Biochemistry 49:2443‐2453. | |
| Internet Resources | |
| There are abundant resources available on the Internet covering software, detailed experimental step‐by‐step protocols, video instructions, and discussion forums. The following is only a small subset facilitating the application of the methods described in the present unit. | |
| General practical tools to help designing AUC experiments, including detailed step‐by‐step protocols for conducting SE and SV experiments, a table with criteria for buffer selection, a grid for SV run conditions with different optical systems, instructions for radial calibration of the interference system, and a tutorial for the test for equilibrium. | |
| https://sedfitsedphat.nibib.nih.gov/tools/Protocols/Forms/AllItems.aspx | |
| A video showing the assembly of cells (Balbo et al., ). | |
| http://www.jove.com/video/1530/assembly‐loading‐and‐alignment‐of‐an‐analytical‐ultracentrifuge‐sample‐cell | |
| SEDFIT and SEDPHAT can be freely downloaded from these sites. All methods discussed above are implemented in SEDFIT and SEDPHAT, unless otherwise stated. | |
| https://sedfitsedphat.nibib.nih.gov/software/default.aspx | |
| Extensive online help system and documentation, including a tutorial for ‘getting started’ in SEDFIT | |
| http://www.analyticalultracentrifugation.com/default.htm | |
| Step‐by‐step examples for the data analysis with c(s). | |
| http://www.analyticalultracentrifugation.com/examples.htm | |
| Getting started’ tools for SEDPHAT. | |
| http://www.analyticalultracentrifugation.com/sedphat/concepts_for_getting_started.htm | |
| Tutorials on special topics of AUC and/or SEDFIT or SEDPHAT analyses. | |
| http://www.analyticalultracentrifugation.com/tutorials.htm | |
| Movies illustrating the mechanism of migration of the reaction boundary. | |
| https://sedfitsedphat.nibib.nih.gov/tools/Reaction%20Boundary%20Movies/Forms/AllItems.aspx | |
| A step‐by‐step protocol for sedimentation equilibrium analyses. | |
| https://sedfitsedphat.nibib.nih.gov/tools/Protocols/SedimentationEquilibrium.pdf | |
| GUSSI download site. | |
| http://biophysics.swmed.edu/MBR/software | |
| SEDNTERP for the calculation of buffer densities and viscosities, as well as protein extinction coefficients and partial specific volumes, at John Philo's Web site. | |
| http://www.jphilo.mailway.com/download.htm | |
| SEDFIT‐L discussion forum. | |
| https://list.nih.gov/cgi‐bin/wa.exe?SUBED1=SEDFIT‐L&A=1 | |
| SEDPHAT‐L discussion forum. | |
| https://list.nih.gov/cgi‐bin/wa.exe?A0=SEDPHAT‐L | |
| RASMB discussion forum. | |
| http://rasmb.bbri.org/cgi‐bin/mailman/listinfo/rasmb | |
| These two Web sites provide current information on upcoming workshops. | |
| https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx | |
| http://www.analyticalultracentrifugation.com/default.htm |
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
