In the last year we have embarked on new collaborations that exploit our novel technique for studying particle size distributions at high concentrations by nonideal sedimentation velocity (SV) analytical ultracentrifugation. One particular area of application is the measurement of proteins under conditions suitable for nuclear magnetic resonance (NMR) structure determination. NMR often requires high molar concentrations, which for proteins may lead to weight concentrations that exceed the dynamic range of most techniques for measuring protein size distributions. However, knowledge of the protein higher-order structure is essential for structural analysis by NMR. Thus, as a pilot application for nonideal SV supplementing NMR, jointly with Dr. Lewis Kay (University of Toronto) we have engaged in the collaborative characterization of the oligomeric distribution a molecular chaperone from E. coli that undergoes a concentration-dependent assembly from trimers to hexamers and dodecamers. We have been able to identify conditions and molecular variants that populate predominantly a single state. In the course of these measurements, we have been able to verify the high precision of modeling nonideal sedimentation of concentrated protein solutions. We have also embarked on a collaborative study of the oligomeric state of the mannan-binding lectin (MBL) protein, which is a pattern recognition molecule crucial for activating the immunological complement system, a first line of defense against pathogens. Dr. Thomas Vorup-Jensen (Aarhus University) has established the importance of multimeric, polyvalent interactions in the targeting of MBL. In collaboration with his laboratory, we have been able to characterize the oligomeric size-distribution of MBL in detail in dilute solution, and were able to assess the contributions of reversible and irreversible oligomerization processes. This sets the stage for further measurements in concentrated physiological media. Extending the measurement of size distributions by nonideal SV from proteins to nanoparticles, we have carried out experiments on gold nanoparticle interactions with serum proteins. In this long-standing collaboration with Dr. Alioscka Sousa (University of Sao Paolo) we were able to raise the protein concentrations sufficiently to detect weak protein binding to gold particles under close to physiological conditions. Such interactions could potentially have significant impact on pharmacokinetic properties of therapeutic nanoparticles. We are currently studying gold particles of different chemical properties and of different sizes. The ability to measure macromolecular size-distribution and to quantify weak transient interactions in concentrated solutions allowed us to revisit the question of the solution state of eye lens crystallin, in collaboration with the laboratory of Dr. Graeme Wistow (NEI). The self-association state of crystallins at the extremely high concentrations in the eye lens is critical for understanding mechanism of cataract, which is the leading cause for blindness worldwide. Using the new methodology of nonideal SV have recently been able to demonstrate weak dimerization of gammaS crystallins, which implies the presence of significant populations of crystallin dimers at physiological concentrations. This has a significant impact on our understanding of the energetic balance that keeps eye lens crystallins soluble. In related work, jointly with the laboratory of Dr. Eugene Shakhnovich (Harvard University) we have embarked on the study of mutant gamma crystallin implicated in cataract formation, with the goal to aid the understanding of the aggregation mechanism and kinetics. Finally, we have continued several long-term collaborations. Jointly with the laboratory of Dr. Lawrence Samelson (NCI) we have recently succeeded in the assembly of a four-molecular signaling complex critical for T-cell activation. In follow-up studies utilizing our multi-method analysis tools we have examined protein variants that lend greater stability to this complex. Similarly, using our biosensor analysis for polydisperse ensembles of binding sites, we have continued the characterization of different antibodies and their role in immunity against hepatitis B virus antigens, in collaboration with Dr. Patrizia Farci (NIAID) in the study of molecular immunology in acute liver failure.
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