Characterization of an associated equilibrium folding intermediate of bovine growth hormone

Brems, David N.; Plaisted, S.M.; Kauffman, Emma; Havel, Henry A.

doi:10.1021/bi00369a030

Cited by 86 publications

(60 citation statements)

References 16 publications

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“…Their three-dimensional structure is that of four-helix bundle with the helices arranged in an antiparallel fashion (1). Previous folding studies of bovine growth hormone (bGH) have demonstrated that under conditions of partial denaturation a stable intermediate is formed that is associated (2)(3)(4). The solubility ofthis species was investigated and was shown to be less soluble than the native or denatured conformations (5).…”

mentioning

confidence: 99%

Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis.

Brems

Plaisted

Havel

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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By using oligonucleotide-directed mutagenesis, Lys-112 of bovine growth hormone (bGH) was changed to leucine, and its resulting effect on folding was studied. Equilibrium denaturation curves for the mutant protein exhibit biphasic or nonsymmetrical transitions by a variety of spectroscopic and hydrodynamic techniques, whereas the wild-type protein at the same concentration exhibits symmetrical transitions. Scheme IThe third helix of bGH is made up of residues 107-128 (1), is amphipathic, and has been shown to be critical to association (3, 5). We have shown that partial denaturation results in the formation of I, which retains -50% helix but has lost most of the tertiary structure (4). We speculated that Ias~O: is stabilized in part by the intermolecular packing between hydrophobic faces of the third helices (3). To further test this model we have extended the hydrophobic face of this amphipathic helix by producing a mutant in which Lys-112 is substituted by leucine. Fig. 1

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mentioning

confidence: 99%

Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis.

Brems

Plaisted

Havel

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…Amphiphilic a-helices can be important structural elements in the folding pathway. For example, a helix composed of residues 110-127 in bovine growth hormone is thought to provide a distinct hydrophobic surface for a long-range intramolecular interaction early in the folding process (22,23 Proc. Natl.…”

Section: Resultsmentioning

confidence: 99%

Analysis of the contribution of an amphiphilic alpha-helix to the structure and to the function of ricin A chain.

Morris

Wool²

1994

Proc. Natl. Acad. Sci. U.S.A.

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The A chain of ricin is a cytotoxic RNA Nglycosidase that inactivates eukaryotic ribosomes. The contribution of the amphiphic helix D, which Is distant from the active site, to the catalysis of the depurination of the adenosine at position 4324 in 28S rRNA has been examined by systematic deletion of amino acids. Two sets of consecutive two-or threeamino acid deletions of the 12 residues in helix D, a total of 20 mutants, were constructed. AU 12 of the amino acids could be deleted in one mutant or another without loss of activity; however, mutations that disrupted the amphiphic of the helix led to inactivation of the enzyme. Thus, the minimum contribution of helix D to the structure of the ricin A chain is to provide hydrophobic and hydrophilic surfaces to shield helix E, which has the active-site residues; moreover, no amino acid side chain in helix D makes a specific contribution to the recognition of the RNA substrate or to catalysis; and, finaliy, phasing of the amino acid deletions can be important to the phenotype of mutants.

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“…At the junction between the productive folding and aggregation pathways, the folding reaction is in kinetic competition with the aggregation of the partially folded species. During folding, certain sites on the surface of the folding intermediates may cause the polypeptide chains to be susceptible to self-association (Zettlmeissl et al, 1979;Brems et al, 1986;Cleland & Wang, 1991).…”

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confidence: 99%

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies

Speed¹,

Morshead²,

Wang³

et al. 1997

Protein Science

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The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (Speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, DjavadiOhaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.

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Characterization of an associated equilibrium folding intermediate of bovine growth hormone

Cited by 86 publications

References 16 publications

Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis.

Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis.

Analysis of the contribution of an amphiphilic alpha-helix to the structure and to the function of ricin A chain.

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies

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