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Research


Protein conformation and dynamics    

The folding and association of proteins arise from a complex interplay of non-covalent interactions involving the protein and water with kinetics that span picoseconds to hours. We seek to answer general questions about the process by which protein chains find specific contacts: What secondary and tertiary contacts are crucial to folding and binding? How disordered is the folded state of a protein? How fast do proteins fold and what are the intrinsic dynamical time scales for folding? What is the role of water and hydrophobicity in guiding the collapse or assembly of structure?

Our approach has been to use 2D IR spectroscopy of the amide vibrations of the polypeptide backbone. The secondary structural sensitivity of amide I (C=O stretch) vibrations and the picosecond time resolution of vibrational spectroscopy makes 2D IR an excellent probe of fast protein dynamics. 2D IR experiments are used in combination with laser temperature jumps to probe protein unfolding and dissociation on nanosecond to millisecond time scales. Isotope labeling provides a strategy for studying site-specific contacts and disorder. These experiments are complimented by accurate structure-based models that allow us to calculate spectra from MD simulations.

  Protein Folding
 
 
Amide I spectral simulations of peptides and proteins    

An atomistic interpretation of protein 2D IR experiments is made possible by models that calculate infrared spectra using structures drawn from molecular dynamics (MD) simulations. These mixed quantum-classical models describe the vibrations of the protein's coupled amide I oscillators. As part of this work, we have recently developed a new empirical model for predicting the amide I vibrational frequencies of individual peptide units. The correlation between experimentally observed frequency shifts and electrostatic values from MD simulations are well described by a remarkably simple Stark shift model, allowing us to predict experimental frequencies to within a few wavenumbers of experiment. This model allows us to quantitatively analyze the hydrogen-bonding environment around an isotope labeled carbonyl. Our current work focuses on developing an equivalent experimentally parameterized model for vibrational coupling constants in polypeptides and proteins.

  Amide I Simulations
 
 
β-hairpin folding

We use β-hairpins as to investigate the role of hydrogen bond contact formation and backbone configurational changes in folding, and to understand the structural heterogeneity of folded states. Hairpins can be synthesized with isotope labels to provide multiple local probes of structure. Our studies of folded TrpZip2 reveal variation in the structure the turn and fraying of ends of the strand, as well as the shifts in population between conformers that occur with increasing temperature. Our T-jump experiments reveal the microsecond exchange between turn conformations.

  Trip Conformers
 
Fast protein folding

Temperature-jump 2D IR spectroscopy enables us to track the secondary structure of proteins as they undergo an unfolding transition. Markov state models and spectral modeling is used to extract a structural view of the protein from 2D IR spectra and understand the folding pathways in small proteins. This allows experiments to be compared directly to the structure and folding rates predicted by MD simulations.

NTL9
 
Molecular recognition and binding

We use the insulin monomer-to-dimer transition as a model system to investigate conformational changes associated with protein-protein interactions. By designing dynamics experiments, we can understand how monomers encounter and recognize one another, how proper registry is found to yield a specific interaction, and what conformational changes are needed for dimer formation. We combine temperature-jump 2D IR spectroscopy with MD simulations of dimer dissociation to reveal the contacts necessary to dimerize, the interplay between secondary structure formation and diffusion, and illuminate the role of desolvation and solvent hydrogen bonding.

insulin
 
 
Conformation and folding of elastin-like peptides

Elastin is the protein of our connective tissue that gives its elastic properties. The unusual mechanical properties of elastomeric proteins have been related to amino acid repeat sequences of the form (XPGXX)n, where X are usually hydrophobic side chains. Many structural proposals have been made for these chains, focusing on the turn propensity of PG, and it has been postulated that hydrophobic hydration is the origin of their restoring force. We are investigating the structure and folding of elastin-like peptides of the sequence (VPGVG)n in order to understand the physical origins of the structure and conformational changes on extension and contraction.

elastin peptide
 
 
Ion transport in membrane proteins  

Membrane proteins are key components to a number of biological functions ranging from homostasis to signaling; however, traditional techniques for studying protein structure are perturbative or poorly suited to membrane proteins. We are developing 2D IR spectroscopy as a tool to probe the secondary and tertiary structure of membrane proteins in situ. This work involves methods to obtain large-scale structure and methods to study ion transport by the KcsA potassium channel.










ion transport