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Journal Issue - Volume 3 Issue 7 (July 1994)

Abstract What role does side‐chain packing play in protein stability and structure? To address this question, we compare a lattice model with side chains (SCM) to a linear lattice model without side chains (LCM). Self‐avoiding configurations are enumerated in 2 and 3 dimensions exhaustively for short chains and by Monte Carlo sampling for chains up to 50 main‐chain monomers long. This comparison shows that (1) side‐chain degrees of...

Abstract It has previously been shown that synthetic peptides corresponding to calcium‐binding sites III (SCIII) and IV (SCIV) from troponin‐C can undergo a calcium‐induced dimerization to form the respective homodimers (Shaw GS, Hodges RS, Sykes BD, 1990, Science 249:280–283; Shaw GS et al., 1992a, J Am Chem Soc 114:6258–6259). In addition, an equimolar mixture of SCIII and SCIV has been shown to form preferentially the SCIII/SCIV heterodimer ...

Abstract The N‐terminal src‐homology 2 domain of the p85α subunit of phosphatidylinositol 3′ kinase (SH2‐N) binds specifically to phosphotyrosine‐containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated βPDGF receptor. A titration of a synthetic 12‐residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2‐N domain was monitored using heteronuclear...

Abstract Solvent‐binding sites were compared in 10 different crystal forms of phage T4 lysozyme that were refined using data from 2.6 Å to 1.7 Å resolution. The sample included 18 crystallographically independent lysozyme molecules. Despite different crystallization conditions, variable crystal contacts, changes due to mutation, and varying attention to solvent during crystallographic refinement, 62% of the 20...

Abstract Heat shock transcription factor (HSF) mediates the activation of heat shock genes by binding to its cognate sites with high affinity and specificity. The high‐affinity binding of HSF is dependent on the formation of an HSF homotrimer, which interacts specifically with the heat shock response element (HSE), comprised of 3 inverted repeats of the 5‐bp sequence NGAAN. In order to investigate the thermodynamic basis of the...

Abstract Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N‐ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (δμ, Sahin‐Tóth M, Kaback HR, 1993, Protein Sci 2:1024–1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315Ψ Cys permease reacts with N‐ethylmaleimide or...

Abstract The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib‐CK) were individually replaced by phenylalanine or cysteine using site‐directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp‐223 is identified as an active‐site residue whose replacement even by phenylalanine resulted in...

Abstract The betabellin target structure consists of 2 32‐residue β sheets packed against each other by hydrophobic interactions. We have designed, chemically synthesized, and biophysically characterized betabellin 14S, a single chain, and betabellin 14D, the disulfide‐bridged double chain. The 32‐residue nongenetic betabellin‐14 chain (HSLTASIkaLTIHVQakTATCQVkaYTVHISE, a = D‐Ala, k = D‐Lys) has a palindromic pattern of polar (p),...

Abstract The primary structure of bovine liver UDP‐glucose dehydrogenase (UDPGDH), a hexameric, NAD+‐linked enzyme, has been determined at the protein level. The 52‐kDa subunits are composed of 468 amino acid residues, with a free N‐terminus and a Ser/Asn microheterogeneity at one position. The sequence shares 29.6% positional identity with GDP‐mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site...

Abstract Amino acid sequences of enzymes that catalyze hydrolysis or phosphorolysis of the N‐glycosidic bond in nucleosides and nucleotides (nucleosidases and phosphoribosyltransferases) were explored using computer methods for database similarity search and multiple alignment. Two new families, each including bacterial and eukaryotic enzymes, were identified. Family I consists of Escherichia coli AMP hydrolase (Amn), uridine phosphorylase...

  • Isolation of N ‐acetyllysine

  • Bernard N. Violand, Michael R. Schlittler, Cory Q. Lawson, James F. Kane, Ned R. Siegel, Christine E. Smith, Eric W. Kolodziej, Kevin L. Duffin
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1002/pro.5560030712 (p 1089-1097)

Abstract Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, ←‐N‐acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed‐phase HPLC (RP‐HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP‐HPLC purified protein were used to identify this altered residue as ←‐N‐acetyllysine....

Abstract The 3‐dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T‐PPase) has been determined by X‐ray diffraction at 2.0 Å resolution and refined to R = 15.3%. The structure consists of an antiparallel closed β‐sheet and 2 α‐helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and...

Abstract Covalent cyclization of peptides is an important tool in structure–function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor‐bound conformation. Previously we described a 2‐step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N‐terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res...

Abstract The thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli. The TS protein was purified by sequential chromatography on Q‐Sepharose and phenyl‐Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a...

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