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Biomolecular Dynamics

Visualizing protein conformational dynamics

Proteins execute a variety of remarkable conformational changes in the course of their function. They fold into unique structures with high fidelity and specifically bind each other with remarkable efficiency. We develop 2D IR spectroscopy as a tool for visualizing the conformational dynamics of proteins during folding and binding. Our approach has three prongs: (1) Ultrafast 2D IR spectra provide information that encode snapshots of molecular structure. (2) IR spectroscopic models use structures drawn from MD simulation to interpret the dynamics in 2D IR spectra. (3) By applying fast T-jumps, we can induce large amplitude conformational dynamics over many decades in time, and track the structural changes with 2D IR. Visualizing these processes allows us to better understand functionally relevant cooperative motions of proteins. We have applied this toolbox to the folding of proteins and peptides, and are focusing our efforts on the dynamics protein–protein interactions and DNA hybridization.


Amide I computational spectroscopy

Our primary protein structural tool is IR spectroscopy of the amide I (C=O stretch) vibrations. These vibrational resonances are sensitive to protein secondary structure and hydrogen-bonding to carbonyls, and can be modeled atomistically. To facilitate interpretation of experimental spectra at the microscopic level, we develop the spectroscopic models that predict experimentally observed IR spectra using structures drawn from molecular dynamics simulation. With the help of rigorous methods for evaluating their accuracy, these tools are approaching a level of quantitative agreement that allows us to refine structures from experimental restraints and characterize intrinsically disordered regions.


Protein–protein recognition and binding

Protein–protein interactions are dynamic events, often involving large conformational changes or folding of disordered regions. We are developing methods to characterize the time-dependent processes of molecular recognition and binding that result in a specific interaction. Our approach uses a combination of amide I 2D IR spectroscopy, temperature-jump experiments, advanced protein synthesis and computational modeling. 2D IR on site-specifically isotope-labeled samples is used to characterize protein structures with ultrafast time resolution. A laser-induced T-jump can be used to thermally dissociate a bound protein complex, and 2D IR can track time-resolved conformational changes on nanosecond to millisecond time scales. Atomistic structures from extensively sampled MD simulations performed by our theoretical collaborators are used to interpret the experiments. By integrating these techniques, we seek to create a detailed molecular description of the dynamics of encounter complexes in protein-protein interactions and use them to refine our understanding of coupled folding-and-binding processes. Current protein–protein binding model systems that we are working on are insulin dimer, barnase–barstar, and RNase S.


Membranes and membrane proteins

The function of membrane proteins is intimately tied to their interactions with the membrane environment. These interactions are complex, spanning many length and timescales. We use 2D IR to probe the fast (<ns), high-amplitude electrostatic fluctuations in the membrane environment, which influence collective motions and slower protein-lipid interactions. To study nanosecond and slower dynamics, we use a fast T-jump to induce a gel-to-fluid lipid phase transition. This provides new insight into not only how phase transitions in biological systems occur, but how structural changes in the membrane translate into structural changes in membrane proteins.


DNA spectroscopy and hybridization

DNA oligonucleotides are commonly assumed to hybridize and melt via a simple two state mechanism, even though they experience large scale structural fluctuations such as fraying and bubbling. These conformational changes play an important role in the recognition and binding of DNA by proteins and other biomolecules. Similar to our protein-protein interaction studies, we study the thermal dehybridization of DNA oligos using 2D IR spectroscopy and T-jump experiments. 2D IR and FTIR spectroscopy allow us to track the four DNA bases independently and observe when they dehybridize, allowing us to study the sequence specificity of the mechanism and how some sequences deviate from two state behavior. We also use T-jump spectroscopy to determine the relevant timescales of the dehybridization process providing further insight into the time-scale and mechanism of their dissociation. To complement our experiments we run coarse-grained MD simulations and are developing a 3D lattice model to help elucidate further details of the hybridization mechanism.

DNA T-jump