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H Robert Guy

Publications and source records attributed to H Robert Guy.

7 recordsLinked to original sources
PubMed2004

STAM: simple transmembrane alignment method.

MOTIVATION: The database of transmembrane protein (TMP) structures is still very small. At the same time, more and more TMP sequences are being determined. Molecular modeling is an interim answer that may bridge the gap between the two databases. The first step in homology modeling is to achieve a good alignment between the target sequences and the template structure. However, since most algorithms to obtain the alignments were constructed with data derived from globular proteins, they perform poorly when applied to TMPs. In our application, we automate the alignment procedure and design it specifically for TMP. We first identify segments likely to form transmembrane alpha-helices. We then apply different sets of criteria for transmembrane and non-transmembrane segments. For example, the penalty for insertion/deletions in the transmembrane segments is much higher than that of a penalty in the loop region. Different substitution matrices are used since the frequencies of occurrence of the various amino acids differ for transmembrane segments and water-soluble domains. RESULTS: This program leads to better models since it does not treat the protein as a single entity with the same properties, but accounts for the different physical properties of the various segments. STAM is the first multisequence alignment program that is directly targeted at transmembrane proteins. AVAILABILITY: Source code and installation package are available on request from the authors. Web access is currently implemented.

Algorithms↗
PubMed2004

A model of voltage gating developed using the KvAP channel crystal structure.

Having inspected the crystal structure of the complete KvAP channel protein, we suspect that the voltage-sensing domain is too distorted to provide reliable information about its native tertiary structure or its interactions with the central pore-forming domain. On the other hand, a second crystal structure of the isolated voltage-sensing domain may well correspond to a native open conformation. We also observe that the paddle model of gating developed from these two structures is inconsistent with many experimental results, and suspect it to be energetically unrealistic. Here we show that the isolated voltage-sensing domain crystal structure can be docked onto the pore domain portion of the full-length KvAP crystal structure in an energetically favorable way to create a model of the open conformation. Using this as a starting point, we have developed rather conventional models of resting and transition conformations based on the helical screw mechanism for the transition from the open to the resting conformation. Our models are consistent with both theoretical considerations and experimental results.

Amino Acid Sequence↗
PubMed2004

Models of the structure and voltage-gating mechanism of the shaker K+ channel.

In the preceding, accompanying article, we present models of the structure and voltage-dependent gating mechanism of the KvAP bacterial K+ channel that are based on three types of evidence: crystal structures of portions of the KvAP protein, theoretical modeling criteria for membrane proteins, and biophysical studies of the properties of native and mutated voltage-gated channels. Most of the latter experiments were performed on the Shaker K+ channel. Some of these data are difficult to relate directly to models of the KvAP channel's structure due to differences in the Shaker and KvAP sequences. We have dealt with this problem by developing new models of the structure and gating mechanism of the transmembrane and extracellular portions of the Shaker channel. These models are consistent with almost all of the biophysical data. In contrast, much of the experimental data are incompatible with the "paddle" model of gating that was proposed when the KvAP crystal structures were first published. The general folding pattern and gating mechanisms of our current models are similar to some of our earlier models of the Shaker channel.

Animals↗
PubMed2003

On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL.

COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. SCIENCE: 282:2220-2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.

Amino Acid Sequence↗
PubMed2002

A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension.

MscL, a bacterial mechanosensitive channel of large conductance, is the first structurally characterized mechanosensor protein. Molecular models of its gating mechanisms are tested here. Disulfide crosslinking shows that M1 transmembrane alpha-helices in MscL of resting Escherichia coli are arranged similarly to those in the crystal structure of MscL from Mycobacterium tuberculosis. An expanded conformation was trapped in osmotically shocked cells by the specific bridging between Cys 20 and Cys 36 of adjacent M1 helices. These bridges stabilized the open channel. Disulfide bonds engineered between the M1 and M2 helices of adjacent subunits (Cys 32-Cys 81) do not prevent channel gating. These findings support gating models in which interactions between M1 and M2 of adjacent subunits remain unaltered while their tilts simultaneously increase. The MscL barrel, therefore, undergoes a large concerted iris-like expansion and flattening when perturbed by membrane tension.

Escherichia coli↗