To explore how distal mutations affect binding sites and in proteins communicate, an ensemble-based model of the native state was used to define energetic connectivities between different structural elements Escherichia coli dihydrofolate reductase. Analysis this protein has allowed us identify two important aspects intramolecular communication. First, within a protein, pair-wise couplings exist that magnitude extent which mutational effects propagate from point origin. These can be...

The activation domains of many transcription factors appear to exist naturally in an unfolded or only partially folded state. This seems be the case for AF1/tau1, major transactivation domain human glucocorticoid receptor. We show here that buffers containing natural osmolyte trimethylamineN-oxide (TMAO), recombinant AF1 folds into more a compact structure, as evidenced by altered fluorescence emission, circular dichroism spectra, and ultracentrifugal analysis. conformational transition is...

Studies of individual domains or subdomains the proteins making up nuclear receptor family have stressed their modular nature. Nevertheless, these receptors function as complete proteins. specific mutations suggest that in holoreceptors, intramolecular domain-domain interactions are important for function, but there is little knowledge concerning interactions. The transcriptional transactivation N-terminal part glucocorticoid (GR) appears to inherent structure. To study its with DNA binding...

Amino acid substitutions at distant sites in the Escherichia coli cyclic AMP receptor protein (CRP) have been shown to affect both nature and magnitude of energetics cooperativity cAMP binding, ranging from negative positive. In addition, binding DNA is concomitantly affected. To correlate effects amino on functional global structural properties CRP, partial specific volume (v̄°), coefficient adiabatic compressibility (βs°), rate amide proton exchange were determined for wild-type eight...

The mosquito-borne West Nile (WNV) and dengue 2 (DEN2V) viruses tick-borne Langat (LGTV) Omsk hemorrhagic fever (OHFV) are arthropod-borne flaviviruses (family Flaviviridae, genus Flavivirus). These quite similar at both the nucleotide amino acid level, yet they very divergent in their biological properties diseases cause. objective of this study was to examine putative receptor-binding domains flaviviruses, envelope (E) protein domain III (D3), which assume structures either as part whole...

Human EXOG (hEXOG) is a 5'-exonuclease that crucial for mitochondrial DNA repair; the enzyme belongs to nonspecific nuclease family includes apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form complex with at 1.81 1.85 Å resolution, respectively. A Wing domain, absent other ββα-Me members, suppresses activity, but confers on hEXOG strong 5'-dsDNA exonuclease activity precisely excises dinucleotide using an intrinsic...

Escherichia coli cAMP receptor protein (CRP) controls more than 20 genes. There are significant differences in the promoter regions these Thus, an elucidation of mechanism CRP action requires knowledge about mode selectivity promoters. An earlier study [Heyduk, T., & Lee, J. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 81, 1744-8] indicates that CRP(cAMP)1 conformer exhibits highest affinity for lac PI site operon. It is conceivable binds with to other sites may not be CRP(cAMP)1. To investigate...

Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844−3852]. Cooperativity cAMP binding CRP the apparent DNA affinity are perturbed [Lin Lee (2002) 41, 11857−11867]. The aim this study is explore effects mutation on surface chemistry define consequences these changes affecting specific sequence recognition by CRP. Furthermore, role interplay between identity...

The urea-induced equilibrium unfolding of the alpha-subunit tryptophan synthase (alphaTS) from Escherichia coli can be described by a four-state model, N right harpoon over left I1 I2 U, involving two highly populated intermediates, and [Gualfetti, P. J., Bilsel, O., Matthews, C. R. (1999) Protein Sci. 8, 1623-1635]. To extend physical characterization these stable forms, apparent radius was measured several techniques. Size-exclusion chromatography (SEC), analytical ultracentrifugation...

Isozymes of pyruvate kinase (PK) expressed in rabbit muscle and kidney show different allosteric kinetics. The only amino acid changes the two isozymes, originating from alternative RNA splicing, occur at a stretch 55 acids C domain near subunit interface. self-correcting distance geometry (SECODG) program DIAMOD was used to calculate homology model these interfacial contacts four helix bundle PK dimer, based on X-ray structure tetrameric [Larsen et al. (1994) Biochemistry 33, 6301−6309]....

The cAMP receptor protein (CRP) of Escherichia coli is a transcription factor. affinity CRP for specific DNA sequence significantly enhanced as consequence the binding allosteric effector, cAMP. hinge region, particularly residues 136 and 138, connecting domains has been proposed to play essential roles in transmitting signals. To probe role residue eight D138 mutants wild-type were tested their ability bind lac26 gallac26 promoter sequences this study. A correlation was established between...

E. coli cyclic AMP receptor protein, CRP, is a modular protein that consists of covalent linkage two common structural domains. To probe the mechanism for intramolecular communications and to define unique properties acquired by linkage, structural, functional cAMP- DNA-binding domains CRP were studied separately as independent polypeptides. The N-terminal cAMP-binding domain (α-CRP), including S-CRP CH-CRP, which generated digestion subtilisin chymotrypsin, respectively, are mainly...

The energetic landscape of the allosteric regulatory mechanism rabbit muscle pyruvate kinase (RMPK) was characterized by isothermal titration calorimetry (ITC). Four novel insights were uncovered. (1) ADP exhibits a dual property. Depending on temperature, can regulate RMPK activity switching enzyme to either R or T state. (2) assumption that ligand binding is state-dependent only correct for PEP but not Phe and ADP. (3) effect pH behavior partly due complex pattern proton release absorption...

Escherichia coli cyclic AMP receptor protein (CRP) is a global transcriptional regulator which controls the expression of many different genes. Although nucleotides can bind to CRP with almost equal affinity, only in presence cAMP could wild-type specific DNA sequences. Molecular genetic studies have identified class mutants, CRP*, either do not require exogenous for activation or be activated by cGMP. Thus, these mutants might aid identifying structural elements that are involved modulation...

Rabbit muscle pyruvate kinase (RMPK) is an important allosteric enzyme of the glycolytic pathway catalyzing a transfer phosphate from phosphoenolpyruvate (PEP) to ADP. The energetic landscape regulatory mechanism RMPK was characterized by isothermal titration calorimetry (ITC) in temperature range 4 45 degrees C. ITC data for binding substrates PEP and ADP, inhibitor Phe, combination ADP Phe were globally analyzed. thermodynamic parameters characterizing linked-multiple-equilibrium system...

Occludin is an essential membrane protein component of cellular tight junctions, participating in both cell-cell adhesion the paracellular space and anchoring junctional complex to cytoskeleton. The latter function accomplished through binding C-terminal cytoplasmic region scaffolding proteins that mediate cytoskeletal actin. We isolated a structural domain from bacterial-expressed human occludin native occludin, extracted epithelial (Madin-Darby canine kidney) or endothelial (human brain)...

Mammalian pyruvate kinase exists in four isoforms with characteristics tuned to specific metabolic requirements of different tissues. All the isoforms, except muscle isoform, exhibit typical allosteric behavior. The case isoform is a conundrum. It inhibited by an inhibitor, Phe, yet it has traditionally not been considered as enzyme. In this series study, energetic landscape rabbit (RMPK) was established. phenomenon inhibition Phe shown be physiological. Furthermore, thermodynamics for...