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Journal Issue - Volume 10 Issue 3 (March 2001)

Abstract N1 is the first residue in an α‐helix. We have measured the contribution of all 20 amino acids to the stability of a small helical peptide CH3CO‐XAAAAQAAAAQAAGY‐NH2 at the N1 position. By substituting every residue into the N1 position, we were able to investigate the stabilizing role of each amino acid in an isolated context. The helix content of each of the 20 peptides was measured by circular dichroism (CD) spectroscopy....

Abstract Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two‐dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in‐gel Endoprotease Lys‐C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in‐gel digestion of individual spots were used to screen human, mouse,...

Abstract A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o‐xylene‐monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited...

Abstract Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating...

Abstract An NMR model is presented for the structure of HMG‐D, one of the Drosophila counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA2 DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high‐resolution structure for the ...

Abstract Previous studies on Escherichia coli aspartate transcarbamoylase (ATCase) demonstrated that active, stable enzyme was formed in vivo from complementing polypeptides of the catalytic (c) chain encoded by gene fragments derived from the pyrBI operon. However, the enzyme lacked the allosteric properties characteristic of wild‐type ATCase. In order to determine whether the loss of homotropic and heterotropic properties was attributable to...

Abstract A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an α helix. All of the variants fold and assemble into dodecameric...

Abstract Nuclear magnetic resonance spectroscopy was used to characterize the solution structure and backbone dynamics of a putative precursor form of ω‐conotoxin MVIIA, a 25‐amino‐acid residue peptide antagonist of voltage‐gated Ca2+ channels. The mature peptide is found in the venom of a fish‐hunting marine snail Conus magus and contains an amidated carboxyl terminus that is generated by oxidative cleavage of a Gly residue. The form examined in ...

Abstract The ClpA, ClpB, and ClpC subfamilies of the Clp/HSP100 ATPases contain a conserved N‐terminal region of ∼150 residues that consists of two approximate sequence repeats. This sequence from the Escherichia coli ClpA enzyme is shown to encode an independent structural domain (the R domain) that is monomeric and ∼40% α‐helical. A ClpA fragment lacking the R domain showed ATP‐dependent oligomerization, protein‐stimulated ATPase activity,...

Abstract The DNA‐repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full‐length protein, the minimal DNA‐binding domain, revealed that one‐third of this molecule was disordered. To better characterize structural features of full‐length XPA, we performed time‐resolved trypsin...

Abstract Glutathione S‐transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium‐tolerant host Escherichia coli strain in the preparation of the protein. 10...

Abstract The TyrR protein of Haemophilus influenzae is a 36‐kD transcription factor whose major function is to control the expression of genes important in the biosynthesis and transport of aromatic amino acids. Using 1H and 15N NMR spectroscopy, we have determined the 3D solution structure of the TyrR C‐terminal DNA‐binding domain (DBD) containing residues from 258 to 318 (TyrR[258–318]). The NMR results show that this segment of TyrR consists...

Abstract CODA, an algorithm for predicting the variable regions in proteins, combines FREAD a knowledge based approach, and PETRA, which constructs the region ab initio. FREAD selects from a database of protein structure fragments with environmentally constrained substitution tables and other rule‐based filters. FREAD was parameterized and tested on over 3000 loops. The average root mean square deviation ranged from 0.78 Å for three...

Abstract Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the...

Abstract Proteins are commonly fused to Escherichia coli maltose‐binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation‐prone proteins. To explain this ...

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