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Journal Issue - Volume 10 Issue 7 (July 2001)

Abstract L‐β‐(Thieno[3,2‐b]pyrrolyl)alanine and L‐β‐(thieno[2,3‐b]pyrrolyl)alanine are mutually isosteric and pharmaceutically active amino acids that mimic tryptophan with the benzene ring in the indole moiety replaced by thiophene. Sulfur as a heteroatom causes physicochemical changes in these tryptophan surrogates that bring about completely new properties not found in the indole moiety. These synthetic amino acids were incorporated into ...

Abstract Homobifunctional chemical cross‐linking reagents are important tools for functional and structural characterization of proteins. Accurate measures of the lengths of these molecules currently are not available, despite their widespread use. Stochastic dynamics calculations now provide quantitative measures of the lengths, and length dispersions, of 32 widely used molecular rulers. Significant differences from published data...

Abstract N2 is the second position in the α‐helix. All 20 amino acids were placed in the N2 position of a synthetic helical peptide (CH3CO‐[AXAAAAKAAAAKAAGY]‐NH2) and the helix content was measured by circular dichroism spectroscopy at 273K. The dependence of peptide helicity on N2 residue identity has been used to determine a free‐energy scale by analysis with a modified Lifson‐Roig helix‐coil theory that includes a parameter for...

Abstract In one of the first studies of isolated intermediates in protein aggregation, we have used circular dichroism and fluorescence spectroscopy to characterize metastable oligomers that are formed in the early steps of β‐lactoglobulin heat aggregation. The intermediates show typical molten globule characteristics (secondary structure content similar to the native and less tight packing of the side chains), in agreement with the...

Abstract Correlated enzymatic conformational fluctuations are shown to contribute to the rate of enhancement achieved during catalysis. Cytidine deaminase serves as a model system. Crystallographic temperature factor data for this enzyme complexed with substrate analog, transition‐state analog, and product are available, thereby establishing a measure of atomic scale conformational fluctuations along the (approximate) reaction...

Abstract The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of kcat/Km for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope...

Abstract In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, ΔCp, that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at...

Abstract The glucose transporter GLUT 1 was isolated from human erythrocytes and reconstituted into endogenous membrane lipids. Results from thermal denaturation studies, using differential scanning calorimetry, indicate that the thermal denaturation temperature of GLUT 1 is significantly lower in the presence of ATP. The lowering of this transition temperature is very dependent on pH. At more acidic pH, ATP has a greater effect of...

Abstract In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR‐like proteins, now known as the IclR family, which can be identified by a conserved C‐terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although purification is straightforward, both the IclR and GclR proteins...

Abstract An elongated version of the de novo designed β‐hairpin peptide, BH8, has allowed us to gain insight into the role of electrostatic interactions in β‐hairpin stability. A Lys–Glu electrostatic pair has been introduced by adding a residue at the beginning and at the end of the N‐terminal and C‐terminal strands, respectively, of the β‐hairpin structure, in both orientations. The two resulting peptides and controls having Ala...

Abstract CaVP is a calcium‐binding protein from amphioxus. It has a modular composition with two domains, but only the two EF‐hand motifs localized in the C‐terminal domain are functional. We recently determined the solution structure of this regulatory half (C‐CaVP) in the Ca2+‐saturated form and characterized the stepwise ion binding. This paper reports the 15N nuclear relaxation rates of the Ca2+‐saturated C‐CaVP, measured at four different...

Abstract The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of α‐lytic protease. The wild‐type α‐lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190→Ala binding pocket mutant has a much broader specificity, actively hydrolyzing...

Abstract Monte Carlo simulations are used to assess the adequacy of the Tanford‐Kirkwood prescription for electrostatic interactions in macromolecules. Within a continuum dielectric framework, the approach accurately describes salt screening of electrostatic interactions for moderately charged systems consistent with common proteins at physiological conditions. The limitations of the Debye‐Hückel theory, which forms the statistical...

Abstract Kinetic properties of the dimeric enzyme dUTPase from Leishmania major were studied using a continuous spectrophotometric method. dUTP was the natural substrate and dUMP and PPi the products of the hydrolysis. The trypanosomatid enzyme exhibited a low Km value for dUTP (2.11 μM), a kcat of 49 s−1, strict Michaelis–Menten kinetics and is a potent catalyst of dUDP hydrolysis, whereas in other dUTPases described, this compound acts as a...

Abstract A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of...

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