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Protein Structure, Dynamics, and Function
Summary: Dorothee Kern uses biophysical analytical techniques to unravel the dynamic personality of enzymes, signaling proteins, and the molecules they affect.
A key feature of life is change over time. In a search for how and why biological processes happen, I study, at the molecular level, changes of atomic coordinates in proteins over time. The ultimate goal is to “visualize” proteins at atomic resolution in real time as they function: enzymes during catalysis, signaling proteins in action, and proteins binding to their partners. To accomplish this, we are using a variety of biophysical methods, including NMR (nuclear magnetic resonance) spectroscopy, x-ray crystallography, molecular dynamics simulations and other computational approaches, single-molecule FRET (fluorescence resonance energy transfer), and classical enzymology. We then want to build the bridge from the microscopic dynamic behavior of individual proteins to the macroscopic dynamic behavior of biological function.
Enzymes in Action
That enzymatic activity requires a precise balance between flexibility and stability is a widely accepted concept. However, key questions remain: How do motions on different timescales relate to each other and contribute to this balance? Have proteins evolved so that substates necessary for activity are preferably accessible?
We have developed approaches that allow quantitative and residue-specific measurements of dynamics in enzymes during catalysis by NMR spectroscopy. Strikingly, we found for several enzymes that the rate-limiting step for the overall turnover is a conformational change and not the chemical step. In other words, nature has “perfectionized” the enzyme to lower the energy barrier for chemistry, but collective motions are the price to be paid.
Moreover, we have recently shown that motions in enzymes are not random but preferentially follow the pathways that create the configuration capable of proficient chemistry. This situation is analogous to protein folding, which is biased to sample only a small portion of the energy landscape. The expansion of the concept of nonrandom sampling of conformational space for efficient biological function—from folding to conformational rearrangements within the folded space—combines both phenomena through the energy landscape. We used a combination of NMR relaxation, x-ray crystallography, single-molecule FRET experiments, and molecular dynamics simulations to characterize the timescale and amplitude of motion. The determined predisposition of enzymes to move in the direction utilized for catalysis may be a key factor for the efficiency of biocatalysts.
We are also investigating the hierarchy in space and time for protein dynamics. By comparing a mesophilic and hyperthermophilic enzyme pair, we identified a linkage between three different “tiers” of dynamic timescales: (1) thermally driven, fast (picosecond), local atomic fluctuations; (2) faster (nanosecond) motions of whole segments; and (3) larger amplitude, collective, slower motions (microsecond to millisecond), the timescale of catalysis. Importantly, we were able to demonstrate that those dynamic features are directly connected to function and stability.
We propose that the presampling of conformational states needed for catalysis and selective binding of substrates to these substates might be a general paradigm of enzyme catalysis. We are currently attacking the next immediate questions: How does a protein move from one energy valley into another, and what are the pathways and the transition states? What are the entropic and enthalpic factors contributing to transition barriers? Can minor conformational substates be predicted from known structures? Can we apply the knowledge gained about physical principles of proteins to design proteins with desired functions? Can we successfully incorporate a dynamic view into rational drug design?
Molecular Mechanism of Phosphorylation-Mediated Signaling
Phosphorylation is a widely used mechanism in signaling. Using response regulators as model systems, we are investigating the molecular mechanism of activation. Response regulators are the dominating signaling molecules in bacteria. Through a combination of NMR structural and dynamic experiments, we found that signaling does not work through a mechanism like a light switch (on, off) but rather through a population-shift mechanism; the active state is already populated to a low percentage before phosphorylation, and phosphorylation shifts this preexisting equilibrium. In other words, this sampling of substates is essential for phosphorylation, shifting the textbook paradigm of phosphorylation inducing a new structure. We are exploring a detailed description of the energy landscape for this signaling protein, including both the inactive/active interconversion and the folding. One key question is how proteins can quickly change among distinct structures without unfolding. We anticipate that lessons learned for this kinase are widely applicable to other kinases.
Mechanism of Signaling Mediated by Prolyl Isomerization
Prolyl isomerases, the enzymes that catalyze the reversible cis/trans isomerization of prolyl peptide bonds, have been implicated to play a crucial role in many biological processes, including cell cycle control, ion channel conductance, cell signaling, neurodegeneration, oncogenesis, transcription, and HIV virulence. However, the molecular mechanisms by which these enzymes affect such a variety of biological processes are poorly understood. How can enzymes that do not break or make bonds but just accelerate a 180° rotation about the peptide bond control cell cycle and tubulin dynamics through the Alzheimer's protein tau? The key is most likely a kinetic control mechanism. This, together with the fact that the physical differences between the cis and trans forms of a prolyl peptide bond are so minute, led us to use NMR spectroscopy to hunt for the answers. We have shown for the first time that CypA can indeed catalyze cis/trans isomerization in a natively folded protein on the HIV capsid protein. We are now investigating a number of the other natural targets of prolyl isomerases, including the Alzheimer's protein tau, with the goal of unraveling how their enzymatic function leads to the observed biological behavior. In a search for new inhibitors, we are characterizing the energy landscapes for human CypA and human Pin1, two of the most important prolyl isomerases.
Last updated: May 6, 2008
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