Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase: probing nucleotide binding by multidimensional NMR

ABSTRACT: The skeletal muscle sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a) mediates muscle relaxation by pumping Ca 2+ from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a

High-Resolution 3D Structure Determination of Kaliotoxin by Solid-State NMR Spectroscopy

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins

SIMS: A Hybrid Method for Rapid Conformational Analysis

Proteins are at the root of many biological functions, often performing complex tasks as the result of large changes in their structure. Describing the exact details of these conformational changes, however, remains a central challenge for computational biology due the enormous computational requirements of the problem. This has engendered the development of a rich variety of useful methods designed to answer specific questions at different levels of spatial, temporal, and energetic resolution. These methods fall largely into two classes: physically accurate, but computationally demanding methods and fast, approximate methods. We introduce here a new hybrid modeling tool, the Structured Intuitive Move Selector (SIMS), designed to bridge the divide between these two classes, while allowing the benefits of both to be seamlessly integrated into a single framework. This is achieved by applying a modern motion planning algorithm, borrowed from the field of robotics, in tandem with a well-established protein modeling library. SIMS can combine precise energy calculations with approximate or specialized conformational sampling routines to produce rapid, yet accurate, analysis of the large-scale conformational variability of protein systems. Several key advancements are shown, including the abstract use of generically defined moves (conformational sampling methods) and an expansive probabilistic conformational exploration. We present three example problems that SIMS is applied to and demonstrate a rapid solution for each. These include the automatic determination of ﾑﾑactiveﾒﾒ residues for the hinge-based system Cyanovirin-N, exploring conformational changes involving long-range coordinated motion between non-sequential residues in Ribose- Binding Protein, and the rapid discovery of a transient conformational state of Maltose-Binding Protein, previously only determined by Molecular Dynamics. For all cases we provide energetic validations using well-established energy fields, demonstrating this framework as a fast and accurate tool for the analysis of a wide range of protein flexibility problems

Structural Biology by NMR: Structure, Dynamics, and Interactions

The function of bio-macromolecules is determined by both their 3D structure and conformational dynamics. These molecules are inherently flexible systems displaying a broad range of dynamics on time-scales from picoseconds to seconds. Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as the method of choice for studying both protein structure and dynamics in solution. Typically, NMR experiments are sensitive both to structural features and to dynamics, and hence the measured data contain information on both. Despite major progress in both experimental approaches and computational methods, obtaining a consistent view of structure and dynamics from experimental NMR data remains a challenge. Molecular dynamics simulations have emerged as an indispensable tool in the analysis of NMR data

Proton magnetic resonance study of the tryptophan residue of streptomyces subtilisin inhibitor

Signals from tryptophan 86 and nearby leucine 53 were identified in the 360 and 400 MHz 1H-NMR spectra of Streptomyces subtilisin inhibitor with the aid of specific deuterium substitution and chemical modification combined with measurements of signal intensity, spin-spin coupling and nuclear Overhauser effect. The identified signals were used to monitor the microenvironment of the tryptophan residue. The local conformation around the tryptophan residue was round to be stable against pH at least up to 11.5 both at 25 and 50°C and against temperature up to 85°C at pH 7

Characterization of deep level defects in thermally annealed Fe‐doped semi‐insulating InP by photoinduced current transient spectroscopy

International audienc

Deep Centers in Undoped Semi-insulating InP

Undoped semi-insulating (SI) InP samples, subjected to one-step and multi-step wafer annealing, and lightly and normally Fe-doped SI InP samples without annealing have been characterized by thermally stimulated current (TSC) spectroscopy. A dominant deep center at 0.63 eV is found in all samples and is undoubtedly due to iron. Two prominent TSC traps, T-b (0.44 eV) and T-d (0.33 eV), found in undoped SI InP, are thought to be related to the phosphorus antisite P-In, and traps at low temperatures, like T-e* (0.19 eV), to the phosphrus vacancy V-P

Deep Centers in Undoped Semi-insulating InP

Undoped semi-insulating (SI) InP samples, subjected to one-step and multi-step wafer annealing, and lightly and normally Fe-doped SI InP samples without annealing have been characterized by thermally stimulated current (TSC) spectroscopy. A dominant deep center at 0.63 eV is found in all samples and is undoubtedly due to iron. Two prominent TSC traps, T-b (0.44 eV) and T-d (0.33 eV), found in undoped SI InP, are thought to be related to the phosphorus antisite P-In, and traps at low temperatures, like T-e* (0.19 eV), to the phosphrus vacancy V-P