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Increasing protein conformational stability by optimizing beta-turn sequence.

ABSTRACT: Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions. However, beta-turn sequences often deviate from preferred proline or preferred glycine. Therefore, our strategy involves replacing non-proline and non-glycine beta-turn residues with preferred proline or preferred glycine residues. Here, we develop guidelines for selecting appropriate mutations, and present results for five mutations (S31P, S42G, S48P, T76P, and Q77G) that significantly increase the conformational stability of RNase Sa. The increases in stability ranged from 0.7 kcal/mol to 1.3 kcal/mol. The strategy was successful in overlapping or isolated beta-turns, at buried (up to 50%) or completely exposed sites, and at relatively flexible or inflexible sites. Considering the significant number of beta-turn residues in every globular protein and the frequent deviation of beta-turn sequences from preferred proline and preferred glycine residues, this simple, efficient strategy will be useful for increasing the conformational stability of proteins.

SUBMITTER: Trevino SR

PROVIDER: S-EPMC2084202 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Increasing protein conformational stability by optimizing beta-turn sequence.

Trevino Saul R SR Schaefer Stephanie S Scholtz J Martin JM Pace C Nick CN

Journal of molecular biology 20070809 1

Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions. However, beta-turn sequences often deviate from preferred proline or preferred glycine. Therefor ...[more]

PMID: 17765922

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Project description:UnlabelledHuman La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a β-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting β-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La β-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein.ImportanceHepatitis C virus is known to infect only humans and chimpanzees under natural conditions. This has prevented the development of a small-animal model, which is important for development of new antiviral drugs. Although a number of human-specific proteins are responsible for this species selectivity and some of these proteins--mostly entry factors--have been identified, full multiplication of the virus in mouse cells is still not possible. In this study, we show that a turn in the human La protein that is responsible for the interaction with the viral RNA is highly specific for the human sequence. Replacement of the corresponding mouse sequence with the human sequence allows the mouse La to behave like its human counterpart and support viral growth in the mouse cell efficiently. This observation, in combination with previously identified cell entry factors, should open up the possibility of creating a mouse model of hepatitis C.

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Increasing protein conformational stability by optimizing beta-turn sequence.

Publications

Increasing protein conformational stability by optimizing beta-turn sequence.

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