Highly flexible proteins present a special challenge for structure determination because they are multi-structured yet not disordered, so their conformational ensembles are essential for understanding function. Because spectroscopic measurements of multiple conformational populations often provide sparse data, experiment selection is a limiting factor in conformational refinement. A molecular simulations- and information-theory based approach to select which experiments best refine conformational ensembles has been developed. This approach was tested on three flexible proteins. For proteins where a clear mechanistic hypothesis exists, experiments that test this hypothesis were systematically identified. When available data did not yield such mechanistic hypotheses, experiments that significantly outperform structure-guided approaches in conformational refinement were identified. This approach offers a particular advantage when refining challenging, underdetermined protein conformational ensembles.

No abstract

Highly flexible proteins present as pecial challenge for structure determination because they are multi-structured yet not disordered, so their conformational ensembles are essential for understanding function. Because spectroscopic measurements of multiple conformational populations often providesparse data, experiment selection is alimiting factor in conformational refinement. Am olecular simulations-and information-theory based approacht os elect whiche xperiments best refine conformational ensembles has been developed. This approachw as tested on three flexible proteins.F or proteins where ac lear mechanistic hypothesis exists,e xperiments that test this hypothesis were systematically identified. When available data did not yield such mechanistic hypotheses, experiments that significantly outperform structure-guided approaches in conformational refinement were identified. This approach offers ap articular advantage when refining challenging,u nderdetermined protein conformational ensembles.

The dynamics of phosphocholine and maltoside micelles, detergents frequently used for membrane protein structure determination, were investigated using electron paramagnetic resonance of spin probes doped into the micelles. Specifically, phosphocholines are frequently used detergents in NMR studies, and maltosides are frequently used in x-ray crystallography structure determination. Beyond the structural and electrostatic differences, this study aimed to determine whether there are differences in the local chain dynamics (i.e., fluidity). The nitroxide probe rotational dynamics in longer chain detergents is more restricted than in shorter chain detergents, and maltoside micelles are more restricted than phosphocholine micelles. Furthermore, the micelle microviscosity can be modulated with mixtures, as demonstrated with mixtures of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate with n-dodecylphosphocholine, n-tetradecylphosphocholine, n-decyl-b-D-maltoside, or n-dodecyl-b-D-maltoside. These results indicate that observed differences in membrane protein stability in these detergents could be due to fluidity in addition to the already determined structural differences.

Cytochrome c (cyt c) displays a striking ability to perform many seemingly disparate functions within the cell. It is responsible for shuttling electrons between protein complexes in the mitochondria to generate ATP and is also a primary signal for apoptosis when improperly localized as a result of an interaction with the mitochondrial lipid cardiolipin (CL). We utilized reverse micelle nuclear magnetic resonance (RM-NMR) in order to investigate these different functional roles of cyt c at atomic resolution. The chemical shifts of RM encapsulated cyt c are essentially identical to the free solution protein, confirming structural fidelity. We have determined the structure of encapsulated cyt c to high resolution (0.45 Å backbone RMSD, 0.92 Å heavy atom RMSD) using standard solution NMR methods. Using pseudo-contact shifts (PCS), we find that the majority of the protein structure does not change significantly upon change in redox state. A subset of residues localized at the heme-exposed face of the protein undergo small structural changes upon change in redox state, localized to the binding site on cyt c for its BC1 complex partner. The interaction of cyt c with CL was investigated by titration of the lipid into the RM encapsulated protein. The confined space effect upon protein encapsulation in the RM allowed for separation and characterization of this peripheral interaction from the subsequent lipid insertion and unfolding of cyt c. These experiments provide the first detailed interface of the initial, largely electrostatic phase of the interaction. 461-Pos Board B241 Prion Proteins and Mechanisms of Interaction with Model Membranes