Dataset Information

Simultaneous structure and dynamics of a membrane protein using REDCRAFT: membrane-bound form of Pf1 coat protein.

ABSTRACT: A strategy for simultaneous study of the structure and internal dynamics of a membrane protein is described using the REDCRAFT algorithm. The membrane-bound form of the Pf1 major coat protein (mbPf1) was used as an example. First, synthetic data is utilized to validate the simultaneous study of structure and dynamics with REDCRAFT using dihedral restraints and backbone N-H RDCs from two different alignments. Subsequently, the validated analysis is applied to experimental data and confirms that REDCRAFT produces meaningful structures from sparse RDC data. Furthermore, simulated data from a two-state jump motion is used to illustrate the necessity for simultaneous consideration of structure and dynamics. Disregarding internal dynamics during the course of structure determination is shown to produce an average-state that is not related to the two intermediate states. During the analysis of RDC data from the dynamic model, REDCRAFT appropriately identifies the region separating the static and dynamic domains of the protein. Finally, analysis of experimental data strongly suggests the existence of internal motion between the amphipathic and the transmembrane helices of the membrane-bound form of the protein. The ability to perform fragmented structure determination of each domain without a priori assumption of the order tensors allows an independent determination of the order tensors, which yields a more comprehensive description of protein structure and dynamics and is particularly relevant to the study of membrane proteins.

SUBMITTER: Shealy P

PROVIDER: S-EPMC3970221 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Simultaneous structure and dynamics of a membrane protein using REDCRAFT: membrane-bound form of Pf1 coat protein.

Shealy Paul P Simin Mikhail M Park Sang Ho SH Opella Stanley J SJ Valafar Homayoun H

Journal of magnetic resonance (San Diego, Calif. : 1997) 20100730 1

A strategy for simultaneous study of the structure and internal dynamics of a membrane protein is described using the REDCRAFT algorithm. The membrane-bound form of the Pf1 major coat protein (mbPf1) was used as an example. First, synthetic data is utilized to validate the simultaneous study of structure and dynamics with REDCRAFT using dihedral restraints and backbone N-H RDCs from two different alignments. Subsequently, the validated analysis is applied to experimental data and confirms that R ...[more]

PMID: 20829084

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Project description:The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160 degrees) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state (1)H/(15)N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.

Project description:Internal dynamics of proteins can play a critical role in the biological function of some proteins. Several well documented instances have been reported such as MBP, DHFR, hTS, DGCR8, and NSP1 of the SARS-CoV family of viruses. Despite the importance of internal dynamics of proteins, there currently are very few approaches that allow for meaningful separation of internal dynamics from structural aspects using experimental data. Here we present a computational approach named REDCRAFT that allows for concurrent characterization of protein structure and dynamics. Here, we have subjected DHFR (PDB-ID 1RX2), a 159-residue protein, to a fictitious, mixed mode model of internal dynamics. In this simulation, DHFR was segmented into 7 regions where 4 of the fragments were fixed with respect to each other, two regions underwent rigid-body dynamics, and one region experienced uncorrelated and melting event. The two dynamical and rigid-body segments experienced an average orientational modification of 7° and 12° respectively. Observable RDC data for backbone C'-N, N-HN, and C'-HN were generated from 102 uniformly sampled frames that described the molecular trajectory. The structure calculation of DHFR with REDCRAFT by using traditional Ramachandran restraint produced a structure with 29 Å of structural difference measured over the backbone atoms (bb-rmsd) over the entire length of the protein and an average bb-rmsd of more than 4.7 Å over each of the dynamical fragments. The same exercise repeated with context-specific dihedral restraints generated by PDBMine produced a structure with bb-rmsd of 21 Å over the entire length of the protein but with bb-rmsd of less than 3 Å over each of the fragments. Finally, utilization of the Dynamic Profile generated by REDCRAFT allowed for the identification of different dynamical regions of the protein and the recovery of individual fragments with bb-rmsd of less than 1 Å. Following the recovery of the fragments, our assembly procedure of domains (larger segments consisting of multiple fragments with a common dynamical profile) correctly assembled the four fragments that are rigid with respect to each other, categorized the two domains that underwent rigid-body dynamics, and identified one dynamical region for which no conserved structure could be defined. In conclusion, our approach was successful in identifying the dynamical domains, recovery of structure where it is meaningful, and relative assembly of the domains when possible.

Dataset Information

Simultaneous structure and dynamics of a membrane protein using REDCRAFT: membrane-bound form of Pf1 coat protein.

Publications

Simultaneous structure and dynamics of a membrane protein using REDCRAFT: membrane-bound form of Pf1 coat protein.

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