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Journal Issue - Volume 10 Issue 6 (June 2001)

Abstract Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T...

Abstract The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X‐ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained...

Abstract The yeast cell adhesion protein α‐agglutinin is expressed on the surface of a free‐living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of α‐agglutinin has a β‐sheet‐rich structure, with only ∼2% α‐helix under native conditions (15–40°C at pH 5.5). This region is predicted to fold into three immunoglobulin‐like domains, and models are...

Abstract Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV1 to KIV10) followed by sequences similar to the kringle V and protease domains of...

Abstract Several post‐translational modifications of lysine residues of lens proteins have been implicated in cataractogenesis. In the present study, the molecular weight of an α‐crystallin isolated from the water‐soluble portion of a cataractous human eye lens indicated that it was a modified αB‐crystallin. Further analysis by mass spectrometry of tryptic digests of this modified protein showed that Lys 92 was modified and that the...

Abstract Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P‐loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P‐loop family,...

Abstract The NANP repeating sequence of the circumsporozoite protein of Plasmodium falciparum was displayed on the surface of fd filamentous bacteriophage as a 12‐residue insert (NANP)3 in the N‐terminal region of the major coat protein (pVIII). The structure of the epitope determined by multidimensional solution NMR spectroscopy of the modified pVIII protein in lipid micelles was shown to be a twofold repeat of an extended and non‐hydrogen‐bonded ...

Abstract The energy landscape of a peptide [Ace‐Lys‐Gln‐Cys‐Arg‐Glu‐Arg‐Ala‐Nme] in explicit water was studied with a multicanonical molecular dynamics simulation, and the AMBER parm96 force field was used for the energy calculation. The peptide was taken from the recognition helix of the DNA‐binding protein, c‐Myb. A rugged energy landscape was obtained, in which the random‐coil conformations were dominant at room temperature. The...

Abstract We have investigated amino acid features that determine secondary structure: (1) the solvent accessibility of each side chain, and (2) the interaction of each side chain with others one to four residues apart. Solvent accessibility is a simple model that distinguishes residue environment. The pairwise interactions represent a simple model of local side chain to side chain interactions. To test the importance of these...

Abstract An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water‐soluble proteins surveyed by Robertson and Murphy occurs at T* ∼283K, independent of the heat denaturation temperature, Tm. This observation...

Abstract We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico‐chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD)...

Abstract The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI...

Abstract The folding of apo‐pseudoazurin, a 123‐residue, predominantly β‐sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far‐ and near‐ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min−1, respectively, at pH 7.0 and at 15°C. This process has an activation enthalpy...

Abstract The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5′‐triphosphate (ddCTP). In this report, we present the details of the 2.3‐Å resolution crystal structures of three...

Abstract Amide hydrogen exchange and mass spectrometry have been used to study the pH‐induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D2O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural...

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