Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

This review describes recent advances in studies on the stabilities of the three-dimensional structures of proteins and on the processes leading to the formation of these structures. The term ‘protein folding’ will be used here to denote the process of the conversion of an open polypeptide chain into the unique three-dimensional conformation of the native protein. Experimental and theoretical aspects of protein folding have been reviewed by anfinsen & Scheraga (1975). In the present article, we emphasize advances made since the writing of that review, together with a brief summary of the background of recent studies.

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“…Hogberg-Raibaud & Goldberg (1977) studied the two tryptic fragments of the /? 2 subunit of E. coli tryptophan synthetase.…”

Section: Processmentioning

confidence: 99%

Protein folding

Némethy

Scheraga

1977

Quart. Rev. Biophys.

296

125

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“…In contrast to β-tryptophan synthase, aaTHEP1 is resistant to proteolytic cleavage by both trypsin and endoproteinase Glu-C (figure 10). Compared to published experiments on the proteolytic cleavage of β-tryptophan synthase [10,11], the experimental conditions were chosen in a way that fragmented β-tryptophan synthase (~30 and ~10 kDa fragments) already were further degraded. Even under these conditions, aaTHEP1 remains stable although it contains 36 (20 × K + 16 × R) possible trypsin and 17 (17 × E) possible endoproteinase Glu-C cleavage sites as predicted from the sequence.…”

Section: Resultsmentioning

confidence: 99%

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

2003

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BackgroundTo identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1).ResultsaaTHEP1 was cloned in E. coli, expressed and purified to homogeneity. At a temperature optimum between 70 and 80°C, aaTHEP1 shows enzymatic activity in hydrolyzing ATP to ADP + Pi with kcat = 5 × 10-3 s-1 and Km = 5.5 × 10-6 M. In addition, the enzyme exhibits GTPase activity (kcat = 9 × 10-3 s-1 and Km= 45 × 10-6 M). aaTHEP1 is inhibited competitively by CTP, UTP, dATP, dGTP, dCTP, and dTTP. As shown by gel filtration, aaTHEP1 in its purified state appears as a monomer. The enzyme is resistant to limited proteolysis suggesting that it consists of a single domain. Although THEP1s are annotated as "predicted nucleotide kinases" we could not confirm such an activity experimentally.ConclusionSince aaTHEP1 is the first member of COG1618 that is characterized biochemically and functional information about one member of a COG may be transferred to the entire COG, we conclude that COG1618 proteins are a family of thermophilic NTPases.

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“…Indeed, results similar to those reported here for AK-HDH I have been observed with /?-galactosidase (four chains of Mr 135 000 each) and the ß2 subunit of tryptophan synthase (two chains of Afr 55 000 each). The monomers of both ß-galactosidase and /32-tryptophan synthase consist of (at least) two compactly folded regions which can be isolated as fragments (Ullmann et al, 1968;Hógberg-Raibaud & Goldberg, 1977a). In the case of ß2tryptophan synthase, it was also shown that the isolated fragments are capable of refolding into a compact nativelike structure; interactions between those independent globular regions are needed for the expression of functional abilities (Hógberg-Railbaud & Goldberg, 1977b).…”

Section: Discussionmentioning

confidence: 99%

Folding of aspartokinase-homoserine dehydrogenase I is dominated by tertiary interactions

Müller¹,

Garel²

1984

Biochemistry

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In the presence of guanidine hydrochloride concentrations above 2 M, aspartokinase-homoserine dehydrogenase I remains sufficiently soluble so that the fluorescence and circular dichroism of the protein can be measured. Both parameters show that, up to 3 M guanidine hydrochloride, the protein exists in a stable folded state which possesses a large amount of secondary structure and buried tryptophan residues. This intermediate species is probably monomeric; it is reversibly unfolded by guanidine hydrochloride concentrations between 3 and 4 M. This folded species is formed rapidly from unfolded protein when the denaturant is diluted out, and this rapid folding step precedes all the reactivation steps described previously. The existence of a stable monomeric and folded intermediate indicates that the tertiary interactions have a major contribution to the stability of the native structure of aspartokinase-homoserine dehydrogenase I. Similar measurements were performed on two complementary nonoverlapping fragments: a kinase fragment corresponding to the N-terminal third and a dehydrogenase fragment corresponding to the C-terminal two-thirds of the polypeptide chain. Both fragments exist in a stable folded state up to 2.5 M guanidine hydrochloride. Both fragments show cooperative unfolding transitions between 2.5 and 4 M denaturant. The stability of the folded state of a given region is about the same in an isolated fragment and in the entire chain of aspartokinase-homoserine dehydrogenase I: indeed, an equimolar mixture of these two fragments and the intact chain would give about the same results. This indicates that folding of the kinase and dehydrogenase regions occurs independent ly with a single subunit of the entire protein.

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Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Cited by 57 publications

References 32 publications

Protein folding

Protein folding

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

Folding of aspartokinase-homoserine dehydrogenase I is dominated by tertiary interactions

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