Research Themes

Protein Dynamics

Protein Interactions

Structural Biology

NMR Methods

Collaborators

 

Protein Dynamics
Our efforts in protein dynamics center on the use of high resolution liquid and solid state NMR spectroscopy to characterize the nature of protein motion and its influence on function. Historically, we have focused on the very fast internal motions that are naturally characterized by classical NMR relaxation phenomena. This has required the development of novel isotopic labeling and multidimensional sampling methods and a variety of analytical strategies. NMR is perhaps the most ideal experimental technique for obtaining comprehensive site resolved (i.e. at atomic resolution) information. NMR relaxation studies have revealed that internal dynamics of proteins are surprisingly rich. We have discovered that motion on the side chains is generally much more varied than on the backbone. We are currently interested in understanding the physical origins and biological significance of these motions. A critical product of these studies is the quantitative interpretation of changes in motion as changes in conformational entropy, a previously experimentally inaccessible quantity.

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Protein Interactions
The interaction of proteins with other macromolecules and small ligands is central to biology. We are attempting to understand the thermodynamic origins of high affinity interactions with a particular goal of filling in a missing piece of the puzzle that is the free energy of binding. Historically, the presence of protein conformational entropy or its role in contributing to the free energy of protein interactions has often been ignored. This is mostly likely due to the difficulty in measuring conformational entropy. We believe that protein conformational entropy is in fact significant and, most importantly, is a variable quantity. Over the past decade we have developed a "dynamical proxy" for conformational entropy with the goal of experimentally measuring its variation during changes in protein functional states. We have largely concentrated on high affinity interactions involving proteins with other proteins or small molecules ligands such as drugs.

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Structural Biology
Our main focus in structural biology is the use of reverse micelle encapsulation to overcome many of the problems presented by large and otherwise difficult proteins to the methods of high resolution liquid NMR spectroscopy. Originally, conceived to overcome the slow tumbling problem presented by large soluble proteins we have more recently been using the basic approach to characterize the structures of integral and anchored membrne proteins. Currently, we are using protein encapsulation to study the structure-function relationships for the myrisolyated proteins recoverin and the HIV matrix protein. We are also employing the KcsA potassium channel as a developmental model for using reverse micelles to solubilize integral membrane proteins. Coming close behind are efforts to use this approach to study the G-protein coupled receptors.

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NMR Methods
We continue our efforts to expand the capabilities of reverse micelle encapsulation for enabling high resolution solution NMR studies of proteins that resist more conventional approaches. Originally, developed the overcome the slow tumbling problem presented by large soluble proteins, it has turned out that the approach is useful in a variety of contexts including integral and anchored membrane proteins, protein hydration & dynamics and the effects of confinement on protein stability, folding, dynamics and function. We have had a long standing interest in application of high pressure NMR to studies in protein biophysics and the recently developed generation of apparatus now allows state-of-the-art NMR at 3.0 kbar (45,000 psi). Finally, we have recently become interested in sparse sampling methods and have focused on radial sampling as a means for both rapid data collection and enhanced S/N.

An exciting new application of solutions of reverse micelles is their apparent ability to assist in overcoming implementation of dynamic nuclear polarization of biopolymers in solution.

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Collaborators
We have a number of on-going interactions with groups at Penn and around the world. Most amplify our natural research themes while others are quite distinct effforts.

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