Protein conformational entropy is not slaved to water.

Conformational entropy can be an important element of the thermodynamics of protein functions such as the binding of ligands. The observed role for conformational entropy in modulating molecular recognition by proteins is in opposition to an often-invoked theory for the interaction of protein molecules with solvent water. The "solvent slaving" model predicts that protein motion is strongly coupled to various aspects of water such as bulk solvent viscosity and local hydration shell dynamics.

Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy.

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side-chain dynamics of the α-helical sensory rhodopsin II and the β-barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl-bearing side-chain motion that is largely independent of membrane mimetic.

Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation.

Recent studies suggest that the fast timescale motion of methyl-bearing side chains may play an important role in mediating protein activity. These motions have been shown to encapsulate the residual conformational entropy of the folded state that can potentially contribute to the energetics of protein function. Here, we provide an overview of how to characterize these motions using nuclear magnetic resonance (NMR) spin relaxation methods.

Improving yields of deuterated, methyl labeled protein by growing in HO.

Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the N-H TROSY and C-H methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E.

Solution NMR investigation of the response of the lactose repressor core domain dimer to hydrostatic pressure.

Previous investigations of the sensitivity of the lac repressor to high-hydrostatic pressure have led to varying conclusions. Here high-pressure solution NMR spectroscopy is used to provide an atomic level view of the pressure induced structural transition of the lactose repressor regulatory domain (LacI* RD) bound to the ligand IPTG. As the pressure is raised from ambient to 3kbar the native state of the protein is converted to a partially unfolded form.

Accurate determination of rates from non-uniformly sampled relaxation data.

The application of non-uniform sampling (NUS) to relaxation experiments traditionally used to characterize the fast internal motion of proteins is quantitatively examined. Experimentally acquired Poisson-gap sampled data reconstructed with iterative soft thresholding are compared to regular sequentially sampled (RSS) data. Using ubiquitin as a model system, it is shown that 25 % sampling is sufficient for the determination of quantitatively accurate relaxation rates.

Optimized expression and purification of biophysical quantities of Lac repressor and Lac repressor regulatory domain.

The recombinant production of Lac repressor (LacI) in Escherichia coli is complicated by its ubiquitous use as a regulatory element in commercially-available expression vectors and host strains. While LacI-regulated expression systems are often used to produce recombinant LacI, the product can be heterogeneous and unsuitable for some studies. Alternative approaches include using unregulated vectors which typically suffer from low yield or vectors with promoters induced by metabolically active sugars which can dilute isotope labels necessary for certain biophysical studies.

Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins.

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule.

Elementary tetrahelical protein design for diverse oxidoreductase functions.

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions.

The effects of alpha-helical structure and cyanylated cysteine on each other.

Beta-thiocyanatoalanine, or cyanylated cysteine, is an artificial amino acid that can be introduced at solvent-exposed cysteine residues in proteins via chemical modification. Its facile post-translational synthesis means that it may find broad use in large protein systems as a probe of site-specific structure and dynamics. The C[triple bond]N stretching vibration of this artificial side chain provides an isolated infrared chromophore.