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Journal Issue - Volume 3 Issue 3 (March 1994)

Abstract Proteolytic dissection of native trp repressor and horse heart cytochrome c has been used to infer some of the steps in the folding pathways of the intact proteins. For both proteins, small fragments are capable of undergoing spontaneous noncovalent association to form subdomains with native‐like secondary and/or tertiary structural features, suggesting that dissection/reassembly may be a general method to gain insight into the...

Abstract The recent development of a whole panoply of multidimensional heteronuclear‐edited and ‐filtered NMR experiments has revolutionized the field of protein structure determination by NMR, making it possible to extend the methodology from the IO‐kDa limit of conventional 2‐dimensional NMR to systems up to potentially 35‐40 kDa. The basic strategy for solving 3‐dimensional structures of larger proteins and protein‐ligand...

Abstract Alignment of homologous amino acid sequences reveals that insertion mutations are fairly common in evolution. Hitherto, the structural consequences of insertion mutations on the surface and in the interior of proteins of known structure have received little attention. We report here the high‐resolution X‐ray crystal structures of 2 site‐directed insertion mutants of staphylococcal nuclease. The structure of the first...

Abstract We have used a synthetic peptide consisting of the first 30 residues of striated muscle α‐tropomyosin, with GlyCys added to the C‐terminus, to investigate the effect of N‐terminal acetylation on the conformation and stability of the N‐terminal domain of the coiled‐coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low α‐helical content (approximately 20%) that was only...

Abstract An important question in protein folding is whether compact substructures or domains are autonomous units of folding and assembly. The protomer of the tetrameric D‐glyceraldehyde‐3‐phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has a complex coenzyme‐binding domain, in which residues 1‐146 form a compact substructure with the last 31 residues (313‐333). Here it is shown that the gene of a single‐chain...

  • Characteristics of a de novo designed protein

  • Toshiki Tanaka, Hiromi Kimura, Mayumi Hayashi, Yosinori Fujiyoshi, Ken‐Ichi Fukuhara, Haruki Nakamura
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1002/pro.5560030306 (p 419-427)

Abstract A series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo. Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host. After BrCN treatment, the protein was purified to homogeneity. The refolded proteins are globular and exist as monomers. One of the designed...

Abstract Thioltransferase (glutaredoxin) was purified from human red blood cells essentially as described previously (Mieyal JJ et al., 1991a, Biochemistry 30:6088–6097). The primary sequence of the HPLC‐pure enzyme was determined by tandem mass spectrometry and found to represent a 105‐amino acid protein of molecular weight 11,688 Da. The physicochemical and catalytic properties of this enzyme are common to the group of proteins...

Abstract We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, α‐chymotrypsin, and β‐lactoglobulin, all of which are rich in β‐sheet, together with that of the model β‐turn peptide L‐pro‐L‐leu‐gly‐NH2. Possible ROA signatures of antiparallel β‐sheet include a strong sharp positive band at ∼ 1,313 cm” associated with backbone amide III CαH and NH deformations, and an amide I couplet, negative...

Abstract Analyses of sequences made available through the Escherichia coli genome project in the 87.2‐89.2‐min and 81.5‐84.5‐min regions have revealed 2 putative operons encoding proteins of the phosphoenolpyruvate: sugar phosphotransferase system (PTS). The first putative operon, designated frv, includes 4 open reading frames (ORFs), ORFf147, ORFf485, ORFf356, and ORFf582. ORFf147 and ORFf485 comprise an Enzyme IIA‐Enzyme IIBC pair of the PTS....

Abstract Interactions between the purified recombinant receptor extracellular domain (RED) of the human low‐affinity neurotrophin receptor (LANR) and recombinant human brain‐derived neurotrophic factor, neurotrophin‐3 (NT‐3) and neurotrophin‐4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the...

Abstract Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a...

Abstract Using site‐directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme‐substrate complex to 1.8 Å resolution using a catalytically...

Abstract In order to understand the nature of ATP and L‐glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site‐directed mutagenesis. Individual mutations (Arg → Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for...

Abstract The structure of many proteins consists of a combination of discrete modules that have been shuffled during evolution. Such modules can frequently be recognized from the analysis of homology. Here we present a systematic analysis of the modular organization of all sequenced proteins. To achieve this we have developed an automatic method to identify protein domains from sequence comparisons. Homologous domains can then be...

Abstract Multiple copy sampling and the bond scaling‐relaxation technique are combined to generate 3‐dimensional conformations of protein loop segments. The computational efficiency and sensitivity to initial loop copy dispersion are analyzed. The multicopy loop modeling method requires approximately 20‐50% of the computational time required by the single‐copy method for the various protein segments tested. An analytical formula is...

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