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Journal Issue - Volume 12 Issue 11 (November 2003)

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength-selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans...

We have used proton magnetic relaxation dispersion (MRD) to study the self-association of bovine -lactoglobulin variant A (BLG-A) as a function of temperature at pH 4.7 (dimer-octamer equilibrium) and as a function of NaCl concentration at pH 2.5 (monomer-dimer equilibrium). The MRD method identifies coexisting oligomers from their rotational correlation times and determines their relative populations from the associated dispersion amplitudes. From MRD-derived correlation times and hydrodynamic...

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their...

The goal of this work is to characterize structurally ambivalent fragments in proteins. We have searched the Protein Data Bank and identified all structurally ambivalent peptides (SAPs) of length five or greater that exist in two different backbone conformations. The SAPs were classified in five distinct categories based on their structure. We propose a novel index that provides a quantitative measure of conformational variability of a sequence fragment. It measures the context-dependent width...

Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was...

Cation- interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation- interactions in a diagonal position of a -hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from -0.20 to -0.48 kcal/mole. Our experimental values for the diagonal cation- interactions are similar to those found in -helical...

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the antibody imprint method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were...

One of the biggest challenges in pharmaceutical research is obtaining integral membrane proteins in a functional, solubilized, and monodisperse state that provides a native-like environment that maintains the spectrum of in vivo activities. Many of these integral membrane proteins are receptors, enzymes, or other macromolecular assemblies that are important drug targets. An example is the general class of proteins composed of seven-transmembrane segments (7-TM) as exemplified by the...

Despite the broad biological importance of G protein-coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS5, consists of the N-terminal and third extracellular loop regions of CC chemokine receptor 3...

We present MASS (Multiple Alignment by Secondary Structures), a novel highly efficient method for structural alignment of multiple protein molecules and detection of common structural motifs. MASS is based on a two-level alignment, using both secondary structure and atomic representation. Utilizing secondary structure information aids in filtering out noisy solutions and achieves efficiency and robustness. Currently, only a few methods are available for addressing the multiple structural...

What determines the shape of the allowed regions in the Ramachandran plot? Although Ramachandran explained these regions in terms of 1-4 hard-sphere repulsions, there are discrepancies with the data where, in particular, the R, L, and -strand regions are diagonal. The R-region also varies along the -helix where it is constrained at the center and the amino terminus but diffuse at the carboxyl terminus. By analyzing a high-resolution database of protein structures, we find that certain 1-4...

A sequence in yeast MAT2/MCM1/DNA complex that folds into -helix or -hairpin depending on the surroundings has been known as chameleon sequence. We obtained the free-energy landscape of this sequence by using a generalized-ensemble method, multicanonical molecular dynamics simulation, to sample the conformational space. The system was expressed with an all-atom model in explicit water, and the initial conformation for the simulation was a random one. The free-energy landscape demonstrated that...

The anti-17-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in...

The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3,5-ADP, 2,5-ADP, 5-ADP, U-2-p and U-3-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5-ADP (Ki = 1.2 M), adopts a syn conformation (in contrast to 3,5-ADP and 2,5-ADP, which adopt an anti), and it is the - rather than the -phosphate group that...

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase (GST ): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST at pH 7.0 and 25°C as assayed using mBBr as...

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