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A refinement protocol to determine structure, topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints.

ABSTRACT: To fully describe the fold space and ultimately the biological function of membrane proteins, it is necessary to determine the specific interactions of the protein with the membrane. This property of membrane proteins that we refer to as structural topology cannot be resolved using X-ray crystallography or solution NMR alone. In this article, we incorporate into XPLOR-NIH a hybrid objective function for membrane protein structure determination that utilizes solution and solid-state NMR restraints, simultaneously defining structure, topology, and depth of insertion. Distance and angular restraints obtained from solution NMR of membrane proteins solubilized in detergent micelles are combined with backbone orientational restraints (chemical shift anisotropy and dipolar couplings) derived from solid-state NMR in aligned lipid bilayers. In addition, a supplementary knowledge-based potential, E (z) (insertion depth potential), is used to ensure the correct positioning of secondary structural elements with respect to a virtual membrane. The hybrid objective function is minimized using a simulated annealing protocol implemented into XPLOR-NIH software for general use.

SUBMITTER: Shi L

PROVIDER: S-EPMC2824793 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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A refinement protocol to determine structure, topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints.

Shi Lei L Traaseth Nathaniel J NJ Verardi Raffaello R Cembran Alessandro A Gao Jiali J Veglia Gianluigi G

Journal of biomolecular NMR 20090714 4

To fully describe the fold space and ultimately the biological function of membrane proteins, it is necessary to determine the specific interactions of the protein with the membrane. This property of membrane proteins that we refer to as structural topology cannot be resolved using X-ray crystallography or solution NMR alone. In this article, we incorporate into XPLOR-NIH a hybrid objective function for membrane protein structure determination that utilizes solution and solid-state NMR restraint ...[more]

PMID: 19597943

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Project description:Cryo-EM has become one of the prime methods for protein structure elucidation, frequently yielding density maps with near-atomic or medium resolution. If protein structures cannot be deduced unambiguously from the density maps, computational structure refinement tools are needed to generate protein structural models. We have previously developed an iterative Rosetta-MDFF protocol that used cryo-EM densities to refine protein structures. Here we show that, in addition to cryo-EM densities, incorporation of other experimental restraints into the Rosetta-MDFF protocol further improved refined structures. We used NMR chemical shift (CS) data integrated with cryo-EM densities in our hybrid protocol in both the Rosetta step and the molecular dynamics (MD) simulations step. In 15 out of 18 cases for all MD rounds, the refinement results obtained when density maps and NMR chemical shift data were used in combination outperformed those of density map-only refinement. Notably, the improvement in refinement was highest when medium and low-resolution density maps were used. With our hybrid method, the RMSDs of final models obtained were always better than the RMSDs obtained by our previous protocol with just density refinement for both medium (6.9 Å) and low (9 Å) resolution maps. For all the six test proteins with medium resolution density maps (6.9 Å), the final refined structure RMSDs were lower for the hybrid method than for the cryo-EM only refinement. The final refined RMSDs were less than 1.5 Å when our hybrid protocol was used with 4 Å density maps. For four out of the six proteins the final RMSDs were even less than 1 Å. This study demonstrates that by using a combination of cryo-EM and NMR restraints, it is possible to refine structures to atomic resolution, outperforming single restraint refinement. This hybrid protocol will be a valuable tool when only low-resolution cryo-EM density data and NMR chemical shift data are available to refine structures.

Project description:Atomic-level structural information on alphaB-Crystallin (alphaB), a prominent member of the small heat-shock protein family, has been a challenge to obtain due its polydisperse oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on an approximately 580-kDa human alphaB assembled from 175-residue 20-kDa subunits. An approximately 100-residue alpha-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different alpha-crystallin domain constructs isolated from alphaB. In vitro, the chaperone-like activities of full-length alphaB and the isolated alpha-crystallin domain are identical. Chemical shifts of the backbone and C(beta) resonances have been obtained for residues 64-162 (alpha-crystallin domain plus part of the C-terminus) in alphaB and the isolated alpha-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six beta-strands in the alpha-crystallin domain. A majority of residues in the alpha-crystallin domain have similar chemical shifts in both solid-state and solution-state, indicating similar structures for the domain in its isolated and oligomeric forms. Sites of intersubunit interaction are identified from chemical shift differences that cluster to specific regions of the alpha-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within alphaB. Evidence for a novel dimerization motif in the human alpha-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) (1)H-(15)N heteronuclear single quantum coherence spectra as a function of pH. The isolated alpha-crystallin domain undergoes a dimer-monomer transition over the pH range 7.5-6.8. This steep pH-dependent switch may be important for alphaB to function optimally (e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia, which is accompanied by acidosis).

Dataset Information

A refinement protocol to determine structure, topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints.

Publications

A refinement protocol to determine structure, topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints.

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