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Dynamics of Macromolecular Assembly 

Contents

• DMA Overview
• Research Emphases
• Collaboration
• Specialized Instrumentation
• Supporting Tools and Resources
• Meet the DMA Staff
• Selected Publications

The Dynamics of Macromolecular Assembly (DMA) group provides collaborative research support in the characterization of proteins, their size and shape in solution, thermodynamics and kinetics of self-association and heterogeneous protein interactions. We develop and apply methodology in the areas of sedimentation equilibrium and sedimentation velocity (analytical ultracentrifugation), surface plasmon resonance biosensing, photon correlation spectroscopy, isothermal titration and differential scanning microcalorimetry, and circular dichroism. The purpose is to elucidate the relationship of protein structure and function.

Research Emphases

• Protein interactions, their stoichiometry, thermodynamics and kinetics
• Determination of size-and-shape distributions of proteins by hydrodynamic methods
• Assembly of multi-protein complexes in solution
• Protein surface binding reactions and kinetics
• Microfluidic sample handling for small volume biosensing and interfacing biosensors with mass spectroscopy
• Reaction-diffusion problems in flows close to surfaces and in the centrifugal field
• Application of Fredholm integral equations and maximum entropy methods in biophysical techniques
• Global modeling methods for sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation

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Collaboration

Examples for biological problems that are being addressed in collaboration with other NIH scientists include:

• Studies of cell surface receptor-ligand interactions
• Multi-protein complexes of adaptor proteins
• Characterization of filamenting proteins
• Protein-nucleic acid interactions
• Antibody-antigen interactions
• Self-association and hetero-association of membrane proteins in detergent solution
• Protein-small molecule binding and nucleic acid - small molecule binding
• Characterization of drug delivery particles

Besides the development and collaborative application of the biophysical methodology for the characterization of proteins, DMA can, in some cases, provide support for protein purification and expression.

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Specialized Instrumentation

• Five analytical ultracentrifuges with absorbance and interference optics (Beckman Optima XL-A and XL-I)
• Three surface plasmon resonance biosensors (Biacore X, Biacore 3000, and Reichert)
• An isothermal titration and a differential scanning microcalorimeters (CSC and Microcal)
• Dynamic light scattering instruments (Protein Solutions and Brookhaven Instruments)
• A Dawn multi-angle light scattering instrument for SEC-MALLS (Wyatt)
• A CD spectrophotometer (Jasco)

For more information about starting a collaborative project, please contact Peter Schuck.

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Supporting Tools and Resources

Software tools, tutorials, and other supporting resources for the biophysical methodology developed in the DMA labs are currently available for:

• Analytical Ultracentrifugation
• Optical Biosensing

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Meet the Staff

Peter Schuck, Ph.D. - Biophysicist, Chief

Patrick Brown, Ph.D. - Research Fellow

Huaying Zhao, Ph.D. - Research Fellow

Andrea Balbo - Staff Biologist

Faezeh Razjouyan - Post-Baccalaureate IRTA

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Selected Publications

P. Schuck "Analytical Ultracentrifugation." In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Springer. (in press) (2006).

Balbo A, Minor KH, Velikovsky CA, Mariuzza RA, Peterson CB, Schuck P "Studying multi-protein complexes by multi-signal sedimentation velocity analytical ultracentrifugation." Proceedings of the National Academy of Sciences 102:81-86 (2005).

Balbo A, Schuck P. "Ultracentrifugation in the analysis of protein-protein interactions." In: Protein-Protein Interactions, Cold Spring Harbor Laboratory Press ( E. Golemis, P.D. Adams, Edts) Cold Spring Harbor, New York (2005).

Budihas SR, Gorshkova I, Gaidamakov S, Wamiru A, Bona MK, Parniak MA, Crouch RJ, McMahon JB, Beutler JA, Le Grice SF, "Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones." Nucleic Acids Res., 2005 Mar 1;33(4):1249-56 (2005).

Burgess BR, Schuck P, Garboczi DN. "Dissection of the merozoite surface protein 3, a representative of plasmodium falciparum surface proteins, reveals an oligomeric and highly elongated molecule." Journal of Biological Chemistry, 280:37236-37245 (2005).

Che MM, Boja ES, Yoon H-Y, Gruschus J, Jaffe H, Stauffer S, Schuck P, Fales HM, Randazzo PA. "Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and arf GAP domains." Cellular Signalling, 17:1276-1288 (2005).

Chen Z, Earl P, Damon I, Zhou Y-H, Yu F, Sebrell A, Emerson S, Americo J, Cohen G, Svitel J, Schuck P, Satterfield W, Moss B, Purcell R. "A chimpanzee monoclonal antibody to vaccinia virus B5R protein protects mice against virulent vaccinia virus and neutralizes vaccinia and smallpox viruses in vitro." Proceedings of the National Academy of Sciences USA (in press) (2005).

Chen Z, Moayeri M, Zhou Y-H, Leppla S, Emerson S, Sebrell A, Yu F, Svitel J, Schuck P, Purcell P. Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen. Journal of Infectious Diseases, (in press) (2005).

D.J. Scott and P. Schuck. "A brief introduction to the analytical ultracentrifugation of proteins for beginners." In: Modern Analytical Ultracentrifugation: Techniques and Methods (D.J. Scott, S.E. Harding, A.J. Rowe, Editors) The Royal Society of Chemistry, Cambridge, (2005).

Dam J, Schuck P. "Sedimentation velocity analysis of heterogeneous protein-protein interactions: Sedimentation coefficient distributions c(s) and asymptotic boundary profiles from Gilbert-Jenkins theory." Biophysical Journal, 89:651-666 (2005).

Dam J, Velikovsky CA, Mariuzza RA, Urbanke C, Schuck P. "Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s)." Biophysical Journal, 89:619-634 (2005).

Gaidamakov SA, Gorshkova II, Schuck P, Haruki M, Yamada H, Cerritelli SM, Crouch RJ. "Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain." Nucleic Acids Research, 33:2166-2175 (2005).

Gonzales JM, Velez M, Jimenez M, Alfonso C, Schuck P, Mingorance J, Vicente M, Minton AP, Rivas G. "The cooperative behavior of E. Coli cell division protein FtsZ assembly involves the preferential cyclization of long single-stranded fibrils." Proceedings of the National Academy of Sciences, USA 102:1895-1900 (2005).

Gupta N, Steenbeke TD, Khazanie P, Censoplano NM, Chung EA, Cruz CC, Chaikin MA, Daucher M, Kottilil S, Mavilio D, Schuck P, Sun P, Rabin PL, Radaev S, Van Ryk D, Cicala C, Arthos JA, Fauci AS. "Targeted lysis of HIV-infected cells by natural killer cells armed and triggered by a recombinant immunoglobulin fusion protein: Implications for immunotherapy." Virology, 332:491-497 (2005).

Heeb MJ, Griffin JH, Schuck P, Xu X. "Protein S multimers and monomers each have different anticoagulant activity." Journal of Thrombosis and Haemostasis, 4:385-391 (2005).

Kornblatt JA, Schuck P. "The influence of temperature on the conformation of canine plasminogen: an analytical ultracentrifugation and dynamic light scattering study." Biochemistry, 44:13122-13131 (2005).

Minor KH, Schar CR, Blouse GE, Shore JD, Lawrence DA, Schuck P, Peterson CB. "A mechanism for assembly of complexes of vitronectin and plasminogen activator inhibitor-1 from sedimentation velocity analysis." Journal of Biological Chemistry, 280:28711-28720 (2005).

Schuck P. "Diffusion-deconvoluted sedimentation coefficient distributions for the analysis of interacting and non-interacting protein mixtures." In: Modern Analytical Ultracentrifugation: Techniques and Methods (D.J. Scott, S.E. Harding, A.J. Rowe, Editors) The Royal Society of Chemistry, Cambridge (2005).

Snyder GA, Torabi-Parizi P, Ford JM, Arthos JA, Schuck P, Colonna M, Sun PD. "The DC-SIGN and DC-SIGNR repeat oligomerizes the receptor and increases gp120 binding affinity." Journal of Virology, 79:4589-4598. (2005).

Vorup-Jensen T, Carman CV, Shimaoka M, Schuck P, Svitel J, Springer TA. "Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin {alpha}X{beta}2." Proceedings of the National Academy of Sciences, USA 102:161401619 (2005).

Yikilmaz E, Rouault TA, Schuck P. "Self-association and ligand-induced conformational changes of iron-regulatory proteins 1 and 2." Biochemistry, 44:8470-8478 (2005).

Boukari H, Nossal R, Sackett DL, Schuck P. "Hydrodynamics of nanoscopic tubulin rings in dilute solution." Physical Review Letters 93:098106 (2004).

Dam J, Schuck P. "Determination of sedimentation coefficient distributions by direct modeling of the sedimentation boundary with Lamm equation solutions." Methods in Enzymology, 384:185-212 (2004).

Gonzales NR, Padlan EA, DePascalis R, Schuck P, Schlom J, Kashmiri SV. "SDR grafting of a murine antibody using multiple human germline templates to minimize its immunogenicity." Molecular Immunology, 41:863-872 (2004).

Guan R, Malchiodi EL, Wang Q, Schuck P, Mariuzza RA. "Crystal structure of the c-terminal peptidoglycan-binding domain of human peptidoglycan recognition protein I-alpha reveals sodium-mediated receptor dimerization." Journal of Biological Chemistry, 279:31873-31882 (2004).

Houtman JCD, Higashimoto Y, Dimasi N, Cho S, Yamaguchi H, Bowden B, Regan C, Malchiodi EL, Mariuzza RA, Schuck P, Appella E, Samelson LE. "Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences." Biochemistry, 43:4170-4178 (2004).

Schuck P "A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co-solutes." Biophysical Chemistry, 108:187-200 (2004).

Schuck P "A model for sedimentation in inhomogeneous media. II. Compressibility of aqueous and organic solvents." Biophysical Chemistry, 108:201-214 (2004).

Schuck, P. "Sample Delivery System with Laminar Mixing for Microvolume Biosensing", Patent Pending (DHHS E-143-2000/0-EP-04).

Tziatzios C, Schubert D, Schuck P, Lancaster CRD, Gennis RB, Barquera B. The state of association of the Na+ -translocating NADH: quinon oxidoreductase in detergent solution. An ultracentrifugation study. Journal of Colloid and Polymer Science, 127:48-53 (2004).

Vistica J, Dam J, Balbo A, Yikilmaz E, Mariuzza RA, Rouault TA, Schuck P. "Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition." Analytical Biochemistry, 326:234-256 (2004).

Ali SA, Iwabuchi N, Matsui T, Hirota K, Kidokoro S, Arai M, Kuwajima K, Schuck P, Arisaka F, "Reversible and fast association of a molecular chaperone, gp57A, of bacteriophage T4." Biophysical Journal 85:2606-2618 (2003).

Andersen JF, Francischetti IMB, Valenzuela JG, Schuck P, Ribeiro JMC. "Inhibition of hemostasis by a high-affinity biogenic amine-binding protein from the saliva of a blood feeding insect." Journal of Biological Chemistry, 278:4611-4617 (2003).

Dam J, Guan R, Natarajan K, Dimasi N, Chlewicki LK, Kranz DM, Schuck P, Margulies DH, Mariuzza RA. "Variable MHC class I engagement by Ly49 NK cell receptors revealed by the crystal structure of Ly49C bound to H-2K b." Nature Immunology, 4:1213-1222 (2003).

DePascalis R, Gonzales NR, Padlan EA, Schuck P, Batra SK, Schlom J, Kashmiri SVS. "In vitro affinity maturation of an SDR-grafted humanized anti-carcinoma antibody (HuCC49V10): Isolation and characterization of minimally immunogenic high affinity variants." Clinical Cancer Research, 9:5521-5531 (2003).

Gonzales NR, Padlan EA, DePascalis R, Schuck P, Schlom J, Kashmiri SVS. "Minimizing immunogenicity of the SDR-grafted humanized antibody CC49 by genetic manipulation of the framework residues." Molecular Immunology, 40:337-349 (2003).

Lebowitz J, Lewis MS, Schuck P. "Back to the Future: A rebuttal to Henryk Eisenberg." Protein Science, 12:2649-2650 (2003).

Rosovitz MJ, Schuck P, Varughese M, Chopra AP, Singh Y, McGinnis LM, Leppla SH. "Alanine scanning mutations in anthrax toxin protective antigen domain 4 reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody." Journal of Biological Chemistry, 278:30936-30944 (2003).

Schuck P "On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation." Analytical Biochemistry, 320:104-124 (2003).

Svitel J, Balbo A, Mariuzza R, Gonzales NE, Schuck P. "Combined affinity and rate constant distributions of analyte or ligand populations from experimental surface-binding kinetics and equilibria." Biophysical Journal, 84:4063-4077 (2003).

Andersen PS, Schuck P, Sundberg EJ, Geisler C, Karjalainen K, Mariuzza RA. "Quantifying the energetics of cooperativity in a ternary protein complex." Biochemistry, 41:5177-5184 (2002).

Arthos J, Cicala C, Steenbeke TD, Chun T-W, Dela Cruz C, Hanback DB, Khazanie P, Nam D, Schuck P, Selig SM, VanRyk D, Chaikin M, Fauci AS. "Efficient inhibition of HIV-1 viral replication by a novel modification of sCD4." Journal of Biological Chemistry, 277:11456-11464 (2002).

De Pascalis R, Iwahashi M, Tamura M, Padlan EA, Gonzales NR, Santos AD, Giuliano M, Schuck P, Schlom J, Kashmiri SVS. "Grafting of abbreviated complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody." Journal of Immunology, 169:3076-3084 (2002).

Gilligan JJ, Schuck P, Yergey AL. "Mass spectrometry after capture and small-volume elution of analyte from a surface plasmon resonance biosensor." Analytical Chemistry, 74:2041-2047 (2002).

Lebowitz J, Lewis MS, Schuck P. "Modern analytical ultracentrifugation in protein science - a tutorial review." Protein Science, 11:2067-2079 (2002).

Perugini MA, Schuck P, Howlett GJ. "Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions." European Journal of Biochemistry, 269:5939-5949 (2002).

Rebois V, Schuck P, Northup J. "Elucidating kinetic and thermodynamic constants for the interaction of G protein subunits and receptors by surface plasmon resonance spectroscopy.(2002) Methods in Enzymology, 344:15-42 (2002).

Schuck P, Perugini MA, Gonzales NR, Howlett GJ, and Schubert D. "Size-distribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems." Biophysical Journal, 82:1096-1111 (2002).

Taraporewala ZF, Schuck P, Ramig RF, Patton JT. "Analysis of a rotavirus temperature-sensitive mutant indicates that NSP2 octamers are the functional form of the protein." Journal of Virology, 76:7082-93 (2002).

Abrantes M, Magone MT, Boyd LF, Schuck P. "Adaptation of a surface plasmon resonance biosensor with microfluidics for use with small sample volumes and long contact times." Analytical Chemistry, 73:2828-2835 (2001).

Center RJ, Schuck P, Leapman RD, Earl PL, Moss B, Arthur LO, Lebowitz J. "Oligomeric structure of soluble and virion-associated forms of the simian immunodeficiency virus envelope proteins in the pre-fusion activated conformation." Proceedings of the National Academy of Sciences, USA 98:14877-14882 (2001).

Li Y, Li H, Dimasi N, McCormick JK, Martin R, Schuck P, Schlievert PM, Mariuzza RA: "Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II." Immunity, 14:93-104 (2001).

Solovyova A, Schuck P, Costenaro L, Ebel C. "Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents." Biophysical Journal, 81:1868-1880 (2001).

Boyington JC, Motyka SA, Schuck P, Brooks AG, Sun PD: "Structure of an Ig-like NK cell receptor in complex with a class I MHC ligand: Peptide recognition and allotype specificity." Nature, 405, 537-543 (2000).

Center RJ, Earl PL, Lebowitz J, Schuck P, Moss B: "The HIV-1 gp120 V2 domain mediates gp41-independent inter-subunit contacts." Journal of Virology, 74:4448-4455 (2000).

Huang K-P, Huang FL, Li J, Schuck P, and McPhie P: "Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3." Biochemistry, 29, 7291-7299 (2000).

Perugini MA, Schuck P, Howlett GJ: "Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid." Journal of Biological Chemistry, 275, 36758-36765 (2000).

Schuck P, Braswell EH: Current Protocols in Immunology (Section 18.8.1 - 18.8.22) (2000) "Measurement of protein interactions by equilibrium ultracentrifugation." Kar SR, Kinsbury JS, Lewis MS, Laue RM, and Schuck P. " Analysis of transport experiments using pseudo-absorbance data." Analytical Biochemistry, 285:135-142 (2000).

Schuck P, Rossmanith P: "Determination of the sedimentation coefficient distribution by least-squares boundary modeling." Biopolymers, 54, 328-341 (2000).

Schuck P, Taraporewala Z, McPhee P, Patton JT: "Rotavirus nonstructural protein NSP2 is an octamer and undergoes ligand-induced conformational changes." Journal of Biological Chemistry, (2000).

Schuck P: "Size distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling." Biophysical Journal, 78, 1606-1619 (2000).

Natarajan K, Boyd LF, Schuck P, Yokoyama WM, Eilat D, Margulies DH: "Interaction of NK cell inhibitory receptor Ly-49A with H-2Dd. Identification of a site distinct from the TCR site." Immunity, 11, 591-601 (1999).

Reiter Y, Schuck P, Boyd LF, and Plaksin D: "Antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules and their characterization." Journal of Molecular Biology, 290, 685-698 (1999)

Schuck P, Boyd LF, Andersen PS: "Measuring protein interactions by optical biosensors." Current Protocols in Protein Science, (Section 20.2.1-20.2.21) (1999).

Schuck P, Demeler B: "Direct sedimentation analysis of interference-optical data in analytical ultracentrifugation." Biophysical Journal, 76, 2288-2296 (1999).

Schuck P, Radu CG, Ward ES: "Sedimentation equilibrium analysis of recombinant mouse FcRn with murine IgG1." Molecular Immunology, 36, 1117-1125 (1999).

Wibbenmeyer JA, Schuck P, Smith-Gill SJ, Willson RC: "Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions" Journal of Biological Chemistry, 274, 26838-26842 (1999).

Schuck P and Millar, DB:. Rapid determination of molar mass in Archibald experiments by direct analysis using the Lamm equation. Analytical Biochemistry, 259:48-53 (1998).

Schuck P: "Sedimentation analysis of non-interacting and self-associating solutes using numerical solutions to the Lamm equation." Biophysical Journal, 75:1503-1512 (1998).

Schuck P, CE McPhee, GJ Howlett. Determination of sedimentation coefficients for small peptides. Biophysical Journal, 74:466-474 (1998).

Lebowitz J, Teale M, Schuck PW: "Analytical band centrifugation of proteins and protein complexes." Biochemical Society Transactions, 26, 745-749 (1998).

Sayers CI, Cain SA, Swan J, Pickett M, Watt PJ, Holgate ST, Padlan EA, Schuck P, Helm BA "Amino acid residues that influence Fc e RI-mediated effector functions of human immunoglobulin E." Biochemistry 37:16152-16164 (1998).

Schuck P, D.B. Millar, and A.A. Kortt: Determination of binding constants by equilibrium titration with circulating sample in a surface plasmon resonance biosensor. Analytical Biochemistry 265:79-91(1998).

Schuck P: "Use of Surface Plasmon Resonance to Probe the Equilibrium and Dynamic Aspects of Interactions Between Biological Macromolecules." Annual Reviews of Biophysics and Biomolecular Structure 26, 541-566 (1997).

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