Extensively Hydrated but Folded: A Novel State of Globular Proteins Stabilized at High Pressure and Low Temperature

AbstractWe studied conformational fluctuations of the transcription factor c-Myb R2 subdomain (52 residues with three Trp) at high pressure and low temperature (5°C) using two different spectroscopic methods, Trp fluorescence and 1H NMR, on its chemically stable mutant C130I (pseudo-wild-type (WTS)), which has a large internal cavity. As pressure was increased from 3 to 300 MPa, the Trp fluorescence λmax of WTS shifted from 342 to ∼355 nm, clearly showing that the three Trp rings become fully exposed to the polar environment, which usually is taken to indicate that the protein underwent unfolding. In contrast, as pressure was increased from 3 to 300 MPa, the high-field-shifted 1H NMR signals characteristic of the folded state showed a still higher-field shift, but no significant changes in their intensity. The last result unequivocally shows that the protein remains largely folded at 300 MPa. The apparent discrepancy between the two predictions would only be solved if one were to postulate the existence of an extensively hydrated but folded state in WTS. Intriguingly, such a state was not found in a cavity-filling mutant of WTS, C130I/V103L, suggesting that this state is mediated by cavity hydration. The generality and significance of this state in proteins are discussed

Molecular analysis of a variant type of familial amyloidotic polyneuropathy showing cerebellar ataxia and pyramidal tract signs

金沢大学がん研究所がん分子細胞制御A Japanese family with atypical type I familial amyloidotic polyneuropathy (FAP) in Iiyama, Japan, was studied. Most of the family members have dysfunctions of the central nervous system, in addition to typical symptoms of type I FAP. The transthyretin (TTR, also called prealbumin) gene of the atypical FAP (FAP-IY) was analyzed with recombinant DNA techniques and a RIA method. FAP-IY was found to have the mutation responsible for the methionine-for-valine substitution at position 30 of TTR, as in the case of typical type I FAP. However, analysis of DNA polymorphisms in the TTR locus showed that FAP-IY has a genetic background differing from that of the typical type I FAP. These observations lead to the consideration that a genetic factor(s) involved in the dysfunction of the central nervous system may locate in a chromosome region in close proximity to the TTR gene

Structure of the porcine LH- and FSH-releasing hormone. II. Confirmation of the proposed structure by conventional sequential analyses

The proposed amino acid sequence of porcine LH- and FSH-releasing hormone (LH-RH/FSH-RH) was reinvestigated by Edman-dansyl degradation after the cleavage of N-terminal pyroglutamyl residue by pyrrolidonecarboxylyl (PCA) peptidase. A C-terminal fragment from chymotrypic digest of LH-RH/FSH-RH was found to be identical to synthetic Gly-Leu-Arg-Pro-Gly-NH 2. The results indicate that the structure initially proposed is correct. The amino acid sequence of porcine LH-RH/FSH-RH is thus (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2

Synthesis of the porcine LH- and FSH-releasing hormone by the solid-phase method

The decapeptide (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2, corresponding to the amino acid sequence of the porcine LH- and FSH-releasing hormone ( LH-RH FSH-RH ) has been synthesized by the solid phase method. After repurification, the synthetic product showed the same physico-chemical and biological properties as natural porcine LH-RH FSH-RH

Further Studies on the Enzymatic and Chemical Inactivation of Hypothalamic FSH-RH

The effect of chemical and enzymatic treatments on the biological activity of porcine follicle-stimulating hormone-releasing hormone (FSH-RH) was studied. FSH-RH activity was not affected by trypsin, pepsin, neuraminidase, carboxypeptidase A, aminopeptidase M and leucine aminopeptidase. However, incubation with chymotrypsin, subtilisin, papain and pyrrolidone carboxylyl peptidase abolished the FSH-RH activity as did treatment with hydrochloric acid (0.9 m, 1 h, 100 °C), diazotized sulfanilic acid, and N-bromosuccinimide. Nitrous acid, sodium metaperiodate, and ninhydrin did not affect the FSH-RH activity appreciably. Amino acid analyses of the purest FSH-RH preparation showed the following composition: His 1, Arg 1, Ser 1, Glu 1, Pro 1, Gly 2, Leu 1, Trp 1, and Tyr 1. These results and information obtained during purification procedures indicate that a basic polypeptide composed of ten amino acids possesses both LH-RH and FSH-RH activity