Sum Frequency Generation and Molecular Dynamics Studies of Biomolecular Orientation at Model Interfaces.

[摘要] Interfaces are ubiquitous in biology, yet few techniques can provide a direct, specific, and in situ means to study the structure of interfacial biomolecules. Recent advances in vibrational spectroscopic techniques are promising, but data analysis and interpretation have been limited by the small number of measurements available to characterize inherently complex systems. In this thesis, Sum Frequency generation Vibrational Spectroscopy and all-atom Molecular Dynamics simulations were combined to create a more detailed picture of molecular orientation of alpha-helical and beta-sheet peptides. SFG was also used to study Gbetagamma and GRK2-Gbetagamma protein complexes at interfaces. The peptide studies focused on the effect of polymer-peptide and peptide-peptide interactions for alpha-helical magainin 2, as well as the effects of conformational flexibility and a non-uniform distribution of molecular orientation for the beta-sheet peptide tachyplesin I. Polarized SFG amide I signals revealed that orientational behavior at the polystyrene/water interface is more complex than may be assumed from CH side chain signals. Both unconstrained simulations and free energy sampling were used to explore the effect of peptide-surface interactions on orientation for magainin 2, and it was found that signal ratios could not be explained by single peptides interacting with the surface in isolation. This orientation may be modulated by the formation of dimers or oligomers. For tachyplesin I, orientation measurements may be affected by the high deformability of the beta-sheet regions.Building from studies on the orientation of simple peptides, a program was developed to facilitate orientation analysis of the multi-subunit GRK2-Gbetagamma complex at a lipid bilayer interface, based on calculations of the net molecular hyperpolarizability of all α-helical segments. It was found that the orientation of GRK2-Gbetagamma cannot be predicted from the crystal structure alone, but does agree with the presence of a recently crystallized receptor binding site. Using a combination of polarized amide I signal ratios and calculated intensity change measurements, it was further demonstrated that Gbetagamma likely reorients in order to bind GRK2. The methods developed in this research should facilitate studies that combine multiple measurements for the study of large proteins and complexes, in situ and in their native environment.

[发布机构] University of Michigan

[效力级别] Molecular Dynamics

[关键词] Sum Frequency Generation;Molecular Dynamics;Biomolecules at Interfaces;Chemistry;Science;Chemistry