Research is proposed to study an interrelated set of problems involving the dynamics and dynamics-structure relationships of proteins, enzymes, enzyme-substrate binding, and the nature of biological water. The proposal is organized into two related parts. The first part discusses protein and enzyme dynamics. The second part involves the properties of biological water and its impact on biological systems. The principal experimental tools are ultrafast 2D-IR vibrational echo spectroscopy and other ultrafast IR methods. The vibrational echo experiments are akin to 2D-NMR except that they directly examine the structural/mechanical degrees of freedom of biological systems on time scales not accessible by other methods. The 2D-IR results are analyzed in conjunction with molecular dynamics simulations and other theoretical approaches. Building on our initial successful work in elucidating the relationship between substrate binding and protein dynamics with 2D-IR spectroscopy, novel approaches will be applied to the important question of how protein structural dynamics are modified by binding of exogenous ligands in the active site. CO and azide probes will be introduced selectively within the active site of several peroxidases. The interplay between key structural motifs and protein function will be examined for several systems. Neuroglobin (Ngb) is a heme protein with a single disulfide bond that is implicated in modulating the protein oxygen binding affinity. Structural and dynamic transformations that occur within Ngb will be probed by biochemically and mutagenically disrupting the disulfide bond. The relationship between protein function and structural transformation will also be examined in other systems such as nitrophorins. Recently developed methodology to introduce site-specific probes of protein dynamics selectively within the active site and at specific locations in the protein will be employed. The effects of nanoscopic confinement on protein unfolding will be probed by studying the denaturation of cytochrome c (cyt c) in aqueous and sol-gel nanopore environments. Denaturation studies with guanidine HCl, urea, methanol, and pH as chemical denaturants will probe the dynamical properties of molten globule states. Biological water differs markedly from bulk water behavior because of the effects of nanoscopic confinement and intimate contact to biological macromolecules. Our successful 2D-IR measurements of the dynamics of nanoscopic water will be extended to reverse micelles with phospholipid and non-ionic surfactants. The dynamics of water at protein interfaces will be determined by confining proteins in the reverse micelles and observing the water hydrogen bond dynamics. Water properties at membrane surfaces play an important role in biological processes because of water's interaction with transmembrane proteins and other biomolecules. The dynamics and interactions of water at the surfaces of model phospholipids membranes will be studied using 2D-IR spectroscopy. The dynamics of water in gramicidin, a model for transmembrane proton channel proteins, in multibilayers will be determined via ultrafast IR spectroscopies.
The structural dynamics of complex biological molecules, such as proteins and enzymes, determine how they perform their biological functions. This project is using advanced infrared laser techniques to directly examine biomolecular structural dynamics and how biomolecule interactions with the surrounding medium, particularly water in biological environments, influence structural dynamics. The methodology builds on previous successful applications and developments of state-of-the-art ultrafast infrared laser experiments.
|Fayer, Michael D (2012) Dynamics of water interacting with interfaces, molecules, and ions. Acc Chem Res 45:3-14|
|Thielges, Megan C; Fayer, Michael D (2012) Protein dynamics studied with ultrafast two-dimensional infrared vibrational echo spectroscopy. Acc Chem Res 45:1866-74|
|Chung, Jean K; Thielges, Megan C; Fayer, Michael D (2012) Conformational dynamics and stability of HP35 studied with 2D IR vibrational echoes. J Am Chem Soc 134:12118-24|
|Rosenfeld, Daniel E; Nishida, Jun; Yan, Chang et al. (2012) Dynamics of Functionalized Surface Molecular Monolayers Studied with Ultrafast Infrared Vibrational Spectroscopy. J Phys Chem C Nanomater Interfaces 116:23428-23440|
|Bagchi, Sayan; Boxer, Steven G; Fayer, Michael D (2012) Ribonuclease S dynamics measured using a nitrile label with 2D IR vibrational echo spectroscopy. J Phys Chem B 116:4034-42|
|Chung, Jean K; Thielges, Megan C; Lynch, Stephen R et al. (2012) Fast dynamics of HP35 for folded and urea-unfolded conditions. J Phys Chem B 116:11024-31|
|Thielges, Megan C; Fayer, Michael D (2011) Time-dependent fifth-order bands in nominally third-order 2D IR vibrational echo spectra. J Phys Chem A 115:9714-23|
|Thielges, Megan C; Chung, Jean K; Axup, Jun Y et al. (2011) Influence of histidine tag attachment on picosecond protein dynamics. Biochemistry 50:5799-805|
|Fenn, Emily E; Wong, Daryl B; Fayer, M D (2011) Water dynamics in small reverse micelles in two solvents: two-dimensional infrared vibrational echoes with two-dimensional background subtraction. J Chem Phys 134:054512|
|Thielges, Megan C; Chung, Jean K; Fayer, Michael D (2011) Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy. J Am Chem Soc 133:3995-4004|
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