Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons.

Mutually Beneficial Combination of Molecular Dynamics Computer Simulations and Scattering Experiments.

We showcase the combination of experimental neutron scattering data and molecular dynamics (MD) simulations for exemplary phospholipid membrane systems. Neutron and X-ray reflectometry and small-angle scattering measurements are determined by the scattering length density profile in real space, but it is not usually possible to retrieve this profile unambiguously from the data alone. MD simulations predict these density profiles, but they require experimental control.

Characterizing Protein-Protein Interactions Using Solution NMR Spectroscopy.

In this chapter, we describe how NMR chemical shift titrations can be used to study the interaction between two proteins with emphasis on mapping the interface of the complex and determining the binding affinity from a quantitative analysis of the experimental data. In particular, we discuss the appearance of NMR spectra in different chemical exchange regimes (fast, intermediate, and slow) and how these regimes affect NMR data analysis.

A Review of the Human Sigma-1 Receptor Structure.

The Sigma-1 Receptor (S1R) is a small, ligand-regulated integral membrane protein involved in cell homeostasis and the cellular stress response. The receptor has a multitude of protein and small molecule interaction partners with therapeutic potential. Newly reported structures of the human S1R in ligand-bound states provides essential insights into small molecule binding in the context of the overall protein structure.

Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor.

The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(Δ35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91-107, which corresponds to the first steroid binding domain-like region.

Characterization of the human sigma-1 receptor chaperone domain structure and binding immunoglobulin protein (BiP) interactions.

The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain.

Characterizing weak protein-protein complexes by NMR residual dipolar couplings.

Protein-protein interactions occur with a wide range of affinities from tight complexes characterized by femtomolar dissociation constants to weak, and more transient, complexes of millimolar affinity. Many of the weak and transiently formed protein-protein complexes have escaped characterization due to the difficulties in obtaining experimental parameters that report on the complexes alone without contributions from the unbound, free proteins. Here, we review recent developments for characterizing the structures of weak protein-protein complexes using nuclear magnetic resonance spectroscopy with special emphasis on the utility of residual dipolar couplings.

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex.

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 muM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners.