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Journal Issue - Volume 9 Issue 2 (2000)

Abstract Genetically‐encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the bis‐arsenical fluorescein dye FlAsH, which specifically recognizes short α‐helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269–212). We find that kinesin tagged with this cysteine‐containing helix binds specifically to FlAsH resin and...

Abstract The crystal structure of ligand‐free tryptophanyl‐tRNA synthetase (TrpRS) was solved at 2.9 Å using a combination of molecular replacement and maximum‐entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl‐5′AMP complex (TAM; Doublie S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17–31). In agreement with small‐angle solution X‐ray scattering...

Abstract Distant homologies between proteins are often discovered only after three‐dimensional structures of both proteins are solved. The sequence divergence for such proteins can be so large that simple comparison of their sequences fails to identify any similarity. New generation of sensitive alignment tools use averaged sequences of entire homologous families (profiles) to detect such homologies. Several algorithms, including...

Abstract Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+‐ascorbate system in acidic environment. Site‐specific mutagenesis was performed at these putative metal‐binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild‐type malic enzyme (WT) were successfully cloned and...

Abstract The relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure‐based thermodynamic approach to study the dissociation of the complex between the toxin microcystin‐LR (MLR) and the catalytic domain of protein...

Abstract The structure of tick anticoagulant peptide (TAP) has been determined by X‐ray crystallography at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP‐BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c‐axis with a diameter of about 45 Å. Individual TAP units interact in a...

Abstract Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., α‐helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such...

Abstract The distance between Ca2+‐binding site III in the C‐terminal domain and Cys35 in the N‐terminal domain in cardiac muscle troponin C (cTnC) was determined with a single‐tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12‐residue binding loop of...

Abstract Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N‐oxide (TMAO). The structure of CspA has five strands of β‐sheet. Strands β1‐β4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all‐or‐none global unfolding mechanism. By contrast, the...

Abstract We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site‐directed spin labeling (SDSL) in combination with isotope enrichment to determine long‐range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold...

Abstract The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the...

Abstract The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F‐G loop, may play an important role in ligand binding and delivery. We studied...

Abstract Electrospray ionization mass spectrometry (ESI‐MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI‐MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N‐terminal domain, containing multiple copies of the repeat...

  • Proline inhibits aggregation during protein refolding

  • Dharmaraj Samuel, Gopal Ganesh, Pey‐Wen Yang, Mei‐Ming Chang, Sue‐Lein Wang, Kuo‐Chu Hwang, Chin Yu, Gurunathan Jayaraman, Thallampuranam Krishnaswamy S. Kumar, Vishwa Deo Trivedi, Ding‐Kwo Chang
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1110/ps.9.2.344 (p 344-352)

Abstract The in vitro refolding of hen egg‐white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive,...

Abstract Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, kcat/Km, for the modified enzyme decreased by a...

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