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Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions, it is indispensable to observe their three‐dimensional (3D) structures at the atomic level in a physiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/baculovirus system using NMR data from living cells exclusively. The method was applied to five proteins, rat calmodulin, human HRas, human ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein G B1 domain. In all cases, we could obtain structural information from well‐resolved in‐cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be a standard tool for protein structure determinations in living eukaryotic cells. For three proteins, we achieved well‐converged 3D structures. Among these, the in‐cell structure of protein G B1 domain was most accurately determined, demonstrating that a helix‐loop region is tilted away from a β‐sheet compared to the conformation in diluted solution. [ABSTRACT FROM AUTHOR]

Proteins are inherently dynamic systems whose motions cover large ranges in both magnitude and timescale. Because of the omnipresence of motion, it is likely that dynamics have important roles in the function of biomolecules. For detailed understanding of a protein's function, the three-dimensional structure and description of its dynamics are therefore required. Structure determination methods are well established, and NMR-relaxation phenomena provide insights into local molecular dynamics; moreover, recently several attempts have been made to detect concerted motion. Here, we present an ensemble-based structure-determination protocol using ensemble-averaged distance restraints obtained from exact NOE rates. Application to the model protein GB3 establishes an ensemble of structures that reveals correlated motion across the ?-sheet, concerted motion between the backbone and side chains localized in the structure core, and a lack of concerted conformational exchange between the ?-sheet and the ?-helix. [ABSTRACT FROM AUTHOR]

Background: The automation of objectively selecting amino acid residue ranges for structure superpositions is important for meaningful and consistent protein structure analyses. So far there is no widely-used standard for choosing these residue ranges for experimentally determined protein structures, where the manual selection of residue ranges or the use of suboptimal criteria remain commonplace. Results: We present an automated and objective method for finding amino acid residue ranges for the superposition and analysis of protein structures, in particular for structure bundles resulting from NMR structure calculations. The method is implemented in an algorithm, CYRANGE, that yields, without protein-specific parameter adjustment, appropriate residue ranges in most commonly occurring situations, including low-precision structure bundles, multi-domain proteins, symmetric multimers, and protein complexes. Residue ranges are chosen to comprise as many residues of a protein domain that increasing their number would lead to a steep rise in the RMSD value. Residue ranges are determined by first clustering residues into domains based on the distance variance matrix, and then refining for each domain the initial choice of residues by excluding residues one by one until the relative decrease of the RMSD value becomes insignificant. A penalty for the opening of gaps favours contiguous residue ranges in order to obtain a result that is as simple as possible, but not simpler. Results are given for a set of 37 proteins and compared with those of commonly used protein structure validation packages. We also provide residue ranges for 6351 NMR structures in the Protein Data Bank. Conclusions: The CYRANGE method is capable of automatically determining residue ranges for the superposition of protein structure bundles for a large variety of protein structures. The method correctly identifies ordered regions. Global structure superpositions based on the CYRANGE residue ranges allow a clear presentation of the structure, and unnecessary small gaps within the selected ranges are absent. In the majority of cases, the residue ranges from CYRANGE contain fewer gaps and cover considerably larger parts of the sequence than those from other methods without significantly increasing the RMSD values. CYRANGE thus provides an objective and automatic method for standardizing the choice of residue ranges for the superposition of protein structures. [ABSTRACT FROM AUTHOR]

Determines the nuclear magnetic resonance (NMR) structure of the monomeric form of the protein gpD. Structural basis of the trimerization during capsid assembly; Mechanism of capsid stabilization by gpD; Statistics for the NMR solution structure of the gpD protein.

The NMR structures of the recombinant cellular form of the prion proteins (PrP9 of the cat (Felis catus), dog (canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented In all of these species. PrV consists of an N-terminal flexibly extended tail with ∼100 amino acid residues and a C-terminal globular domain of ∼100 residues with three a-helices and a short antiparallel β-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrV structures, there are local differences between the globular domains of the different species. Because the five newly determined PrPC structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrPC three- dimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies. [ABSTRACT FROM AUTHOR]

Proteins and nucleic acids maintain the crowded interior of a living cell and can reach concentrations in the order of 200-400 g/L which affects the physicochemical parameters of the environment, such as viscosity and hydrodynamic as well as nonspecific strong repulsive and weak attractive interactions. Dynamics, structure, and activity of macromolecules were demonstrated to be affected by these parameters. However, it remains controversially debated, which of these factors are the dominant cause for the observed alterations in vivo. In this study we investigated the globular folded peptidyl-prolyl isomerase Pin1 in Xenopus laevis oocytes and in native-like crowded oocyte extract by in-cell NMR spectroscopy. We show that active Pin1 is driven into nonspecific weak attractive interactions with intracellular proteins prior to substrate recognition. The substrate recognition site of Pin1 performs specific and nonspecific attractive interactions. Phosphorylation of the WW domain at Ser16 by PKA abrogates both substrate recognition and the nonspecific interactions with the endogenous proteins. Our results validate the hypothesis formulated by McConkey that the majority of globular folded proteins with surface charge properties close to neutral under physiological conditions reside in macromolecular complexes with other sticky proteins due to molecular crowding. In addition, we demonstrate that commonly used synthetic crowding agents like Ficoll 70 are not suitable to mimic the intracellular environment due to their incapability to simulate biologically important weak attractive interactions. [ABSTRACT FROM AUTHOR]