Expression, Folding, and Thermodynamic Properties of the Bovine Oxytocin−Neurophysin Precursor:  Relationships to the Intermolecular Oxytocin−Neurophysin Complex

Eubanks, Sharon; Lu, Min; Peyton, David H.; Breslow, Esther

doi:10.1021/bi9912950

Cited by 8 publications

(37 citation statements)

References 35 publications

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“…The preparation of recombinant WT mature BNP-I and oxytocin precursor and mutagenesis of wild type cDNA have been described previously (17,20). The recombinant mature mutant proteins in the present study were expressed in Escherichia coli, folded at pH 8.0 in the presence of ␤-mercaptoethanol and 10 mM Phe-Tyr-NH 2 , and purified by affinity chromatography as described for the recombinant mature WT protein (17).…”

Section: Preparation Of Wild Type and Recombinant Proteins-bnp-imentioning

confidence: 99%

“…The recombinant mature mutant proteins in the present study were expressed in Escherichia coli, folded at pH 8.0 in the presence of ␤-mercaptoethanol and 10 mM Phe-Tyr-NH 2 , and purified by affinity chromatography as described for the recombinant mature WT protein (17). A large fraction of the protein so prepared is covalently damaged prior to folding (17,20), and the remaining protein folds with variable efficiency depending on its structure (see below). The covalent structures of mutants were confirmed by DNA sequencing of the plasmids from which they were prepared and by mass spectrometry.…”

Section: Preparation Of Wild Type and Recombinant Proteins-bnp-imentioning

confidence: 99%

“…The covalent structures of mutants were confirmed by DNA sequencing of the plasmids from which they were prepared and by mass spectrometry. With proteins that fold efficiently, the bindingcompetent and binding-incompetent proteins are distinguished by clear differences in mass, reflecting the covalent damage of the bindingincompetent protein (20). This was not the case for several of the mutants, indicating the inability of undamaged protein to fold efficiently ("Results").…”

Section: Preparation Of Wild Type and Recombinant Proteins-bnp-imentioning

confidence: 99%

“…The precursor was folded, and the folded precursor was purified essentially as described previously for the WT precursor (20).…”

Section: Preparation Of Wild Type and Recombinant Proteins-bnp-imentioning

confidence: 99%

“…It is also relevant that the present study emphasizes the effects of mutation on mature NP, as opposed to the NP segment of the precursor. We selected this route because of the inefficiency associated with the in vitro folding of the wild type precursor (20), which contrasts with the efficiency of folding the wild type processed protein using added dipeptide to drive the folding process (16,21). In the single case in which the effects of mutation on both the processed protein and precursor are compared, the same effect is seen.…”

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Effects of Diabetes Insipidus Mutations on Neurophysin Folding and Function

Eubanks¹,

Nguyen²,

Deeb³

et al. 2001

Journal of Biological Chemistry

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Mechanisms underlying the pathogenicity of diabetes insipidus mutations were probed by studying their effects on the properties of bovine oxytocin-related neurophysin. The mutations G17V, ⌬E47, G57S, G57R, and C67STOP were each shown to have structural consequences that would diminish the conformational stability and folding efficiency of the precursors in which they were incorporated, and factors contributing to the origins of these property changes were identified. Effects of the mutations on dimerization of the folded proteins were similarly analyzed. The projected relative impact of the above mutations on precursor folding properties qualitatively parallels the reported relative severity of their effects on the biological handling of the human vasopressin precursor, but quantitative differences between thermodynamic effects and biological impact are noted and explored. The sole mutation for which no clear thermodynamic basis was found for its pathogenicity was 87STOP, suggesting that the region of the precursor deleted by this mutation plays a role in targeting independent from effects on folding, or participates in stabilizing interactions unique to the human vasopressin precursor.The hormone vasopressin is synthesized as part of a larger precursor that contains the vasopressin sequence at its amino terminus, followed by that of the disulfide-rich protein neurophysin (NP) 1 and a carboxyl-terminal glycopeptide known as copeptin (Ref. 1 and reviewed in Ref. 2). Oxytocin biosynthesis is similar (3), with the exception that the precursor lacks the copeptin segment, the function of which is unclear (4). The NP components of the two precursors ( VP NP) and ( Oxy NP) are highly homologous; the two bovine neurophysins have almost identical properties in vitro, including similar affinities for each of the two hormones (e.g. Ref.2). Following processing, which cleaves the hormones from NP, the mature hormones remain noncovalently bound to NP by forces basically analogous to those pre-existing within the precursor, leading to analogous NP self-association properties (5). The structure of NP, the nature of the noncovalent interactions between hormones and NP, and the effects of these interactions on NP properties have been extensively investigated in solution (e.g. Ref.2) and by crystallographic analysis (6, 7). Moreover, the central role of NP in vasopressin elaboration has become evident by the demonstration that familial neurogenic diabetes insipidus (FNDI), an autosomal dominant disease characterized by vasopressin deficiency, appears most frequently to be due to mutations in NP (e.g. Refs. 8 and 9) accompanied by loss of the proper targeting of the hormone to regulated neurosecretory granules (e.g. Refs. 10 and 11); retention of the mutated precursor in the endoplasmic reticulum is generally considered the cause of the ultimate death of the affected neurons (10).Many of the mutations involved in diabetes insipidus can be predicted, on the basis of what is already known, to exert their effects by directly or indir...

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Section: Preparation Of Wild Type and Recombinant Proteins-bnp-imentioning

confidence: 99%