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An investigation of structure and stability of DJ-1, a homodimeric protein with proposed antioxidant and chaperone activity was carried out to determine the impact of pathogenic substitutions (M261 and E64D) and over-oxidation with [H.sub.2][O.sub.2]. The results provide a structural basis for DJ-1 dysfunction in familial and sporadic Parkinson's disease (PD), which suggest that dimer stabilization is a reasonable therapeutic strategy to treat both forms of disorder.

A combined N- and C-terminal truncation variant of human apolipoprotein A-I (apoA-I) is designed, expressed in Escherichia coli, isolated and characterized. The results suggest that, in the absence of the C-terminal residues 187-243, removal of residues 1-43 does not induce a major structural reorganization while retaining a protein fold that is consistent with a globular amphipathic alpha-helix bundle.

Research has been conducted on the truncation mutant apolipophorin III of Galleria mellonella. The effect of the C-terminal helices on the apolipophorin III function and structure has been investigated via the design of 3-helix mutants and the details are reported.

Research has been conducted on the Min proteins involved in the bacteria cell division site determination. The role of minD from the gram-negative coccus Neisseria gonorrhoeae min operon has been investigated and discussed.

Results show that the regulatory domain of human cardiac troponin C average heat capacity is 6 calories per mole of potassium and the average heat capacity of calcium binding site 1 is greater than site 2 by 1.3 plus or minus 0.8 calories per mol of potassium, decreasing conformational entropy.

Research has been conducted on the Locusta migratoria apolipoprotein III. The structural reorganization of its amphipathic alpha-helices has been investigated.

Results reveal that the C-terminal deletion mutant of insect apolipophorin III, consisting of three N-terminal helical segments, adopts a alpha-helical secondary structure retaining intact protein characteristics. Data further show that the mutant protein transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes that bind human low-density lipoprotein.

Research reveals that pig heart CoA transferase exists in two oligomeric forms, homodimer and a homotetramer. Data further show that this enzyme duality is observed only under denaturing conditions and under normal physiological conditions, significant kinetic barrier exists that prevents enzyme interconversion.

Research has been conducted on the Manduca sexta apolipophorin III. Fluorescence characteristics of N-(1-pyrene)maleimide have been investigated.

Plasmid-encoded protein FinO suppresses the conjugative transfer of F-like plasmids in Escherichia coli by preventing the translation of the positive activator of transcription of genes called TraJ which is necessary in conjugation. Binding of FinP, a traJ antisense RNA, can also prevent degradation and increases the process of hybridization. Proteolysis, electrophoretic mobility shift assay and circular dichroism spectroscopy were used to identify the regions within FinO that call for interactions with RNA.

Research was conducted to examine the role of Asn and Leu in ice binding and the possibility of designing better ice-binding motifs. A systematic series of seven variant peptides based on the 37-amino acid long HPLC-6 and its two LTAAN ice-binding motifs (IBMs) were synthesized to compare various IBMs without complications caused by differences in peptide length or numbers of IBMs. Results indicate primary roles for Leu and Asn in enhancing antifreeze protein solubility and preventing peptide aggregation.

A study was conducted to analyze various conformational states of chicken apolipoprotein A-I (apoA-I). Spectroscopic methods and near-ultraviolet circular dichroism studies were performed to carry out the analysis. The Jasco J-720 spectrapolarimeter was utilized to determine circular dichroism measurements. Results indicated that chicken apoA-I supports a superficial relationship with the high-density lipoprotein phospholipid monolayer.

The influence of translational and rotational entropy in the unfolding of a dimeric coiled-coil has been investigated in aqueous solution. To this end, the calorimetrically measured unfolding entropies of a synthetic two-stranded alpha-helical coiled-coil with and without a disulfide bond between the two polypeptide chains were compared to determine the entropy of intersubunit cross-linking of a compound. The resultant translational and rotational entropy was found to be more than an order of magnitude smaller than estimates based on ideal gas statistics.

The characterization and design of the disulfide-bridged two-stranded coiled-coil peptide Gla2Nx was performed primarily using the minimalistic approach to create a better understanding of protein sequence. The de novo designed coiled-coil peptide goes through a random coil to alpha-helical transition at pH 7 and 20 degrees C upon metal binding to two sites engineered into the molecule. Results support the conclusion that binding is cooperative since it demonstrated no evidence for the existence of a singly bound intermediate.

Circular dichroism, ultracentrifugation and nuclear magnetic resonance studies were utilized to investigate the influence of the amino acid Thr in type I antifreeze protein in the ice binding. The two central Thr were replaced by Ser and Val to retain the hydroxyl and the methyl group, respectively. It was found that hydrogen bonding has negligible effect on the ice binding, while the entropic effects mediate the binding of antifreeze protein to ice.

Comparative study on the synthesis of the wild-type, recombinant enzyme in E. coli and a hinge-mutant form in which the N- and C-domains are assembled as separate peptides was described. It sought to determine the independent folding of the N- and C-domains of CoA transferase extracted from porcine heart. Results have shown that the productive interaction between the N- and C-domains involves a conformational arrangement favoring proper folding and assembly of CoA transferase.

The kinetics underlying the formation and dissociation of a de novo designed heterodimeric coiled-coil protein consisting of D and K peptides (E/K coil) were investigated using surface plasmon resonance. The experiment, which was conducted to validate the SPR technique, revealed the existence of the linker C-nL-G-G-G where nL = norleucine at the N-terminus of the K peptide and the C-terminus of the E peptide.

Lactam bridges spaced i to i+4, formed between the side chains of Glu3 and Lys7, and Glu10 and Lys 14, stabilize the helical conformation in a 14 amino acid, synthetic peptide. Helical peptides are formed by decreasing the hydrophobicity by amino acid substitution. Lactam bridges placed near the termini of the peptide cause dimerization by negating end effects and presenting a nonpolar face for hydrophobic interaction. Thermal denaturation studies show that lactum based peptides are stable. NMR studies confirm its helical structure.

Spectroscopic and lipid binding studies on the amino and carboxyl terminal fragments of Locusta migratoria apolipophorin III (apoLp-III) show that, besides amphipathic alpha-helices, structural elements of the intact protein helps in the lipid binding abilities of apoLp-III. The isolated amino and carboxyl terminal fragments of the protein cannot imitate the functional capacity of the intact protein, though it can enable interactions with lipids. Ionic and hydrophobic interactions have been suggested to play a main role in the initiation of binding events.

Heteronuclear multidimensional NMR spectroscopic analysis of the binding of Ca2+ to the chicken troponin C recombinant regulatory N-domain shows that the F29W mutation has changed the Ca2+ binding and CD spectroscopic characteristics of the N-domain. The binding of the Ca2+ ion to the NTnC sites I and II takes place in steps with an affinity of 8-10 times more for one site and the chemical shift takes place throughout the N-domain. The experimental procedures of the analysis are discussed.

Spectroscopic methods help analyze the influence of chain length on the stability and the capacity of alpha-helical chain formation of polypeptides in the presence and absence of agents, which induce the alpha-helix. An increase in length of the chain leads to enhanced helicity and stability of the peptides, while the coiled-coil stability is not increased. Hence, the folding of peptides to result in coiled coils is influenced by peptide chain length.

Analysis of the structural and binding properties of the C-terminus, 4K peptide of apolipophorin III (apoLp-III), an apolipoprotein from Manduca sexta, helps study the interaction between apoLp-III lipoprotein membrane and phospholipids. The 4K peptide lacks the ability to bind to lipoprotein surfaces and to convert phospholipid bilayers to discs, indicating that other regions in the molecule contribute to the interaction with lipoproteins and phospholipid bilayers. However, the 4K peptide is capable of forming an amphipathic helix.

Circular dichroism studies reveal that surface-exposed FlaA protein segment of polar flagellar filament of Campylobacter coli predominantly contributes to the serospecific nature of VC167 T2 flagellar filament. The flaA flagellin of C. coli is among the largest of the prokaryotic flagellins and the serospecificity of this flagellin is due to posttranslational modification. The protease-resistance domain of the flagellar filament is positioned between protease-sensitive amino and carboxy terminal domains.

Surface and solution properties of the human plasma lipid transfer protein, cholesteryl ester transfer protein, were examined. Sedimentation equilibrium revealed the monomeric state of the protein in aqueous solution up to 0.62 g/L, or 11.7 micrometers. The presence of apolipoprotein A-I had no effect on the bond between the surface of lipid microemulsion of diameter 26 nm and the protein monomer. The collapsing pressure of the LTP molecules was measured.

Analysis of backbone characteristics of phosphopeptide-complexed and free Src homology 2 domain reveals the link between large-scale restriction of picosecond-time-scale dynamics and peptide binding, caused by protein-protein interactions. NMR data reveal several cross peaks of protein residues in the binding site of SH2 complex.

Hydrodynamic and spectroscopic techniques are employed to determine the properties of the calponin-caltropin complex. Circular dichroism (CD) spectrum shows a secondary structural change in the protein reactants, a decrease in alpha-helix and an increase in beta-sheet. CD is used to monitor guanidine hydrochloride and temperature dependent complex denaturation. Calponin-caltropin can function as a regulatory complex in smooth muscle contraction, and the complex formation is monitored by fluorescence studies.

Study of the orientation of alpha-helical chains in the formation of double-stranded anti-parallel and parallel coiled-coils reveals the involvement of interchain electrostatic reactions. The study involved the designing of four disulfide-bridged coiled-coils. The air oxidation of two 35-residue peptides with the suitable heptad repeat yields these coils.

The structure and stability of Locusta migratoria apolipophorin III (apoLp-III) is affected by deglycosylation and complexation with lipids. The complexation of apoLp-III increased stability and deglycosylation decreased stability. apoLp-III is resistant to pH-induced denaturation. The spectra of apoLp-III shows two peaks corresponding to phenylalanine and tryptophan residues indicating structure change complexation of apoLp-III.

The spectral characteristics of Phe-154 to Trp mutant (F154W) of chicken troponin C (TrC) are influenced by troponin I inhibitory peptide and calcium and magnesium ions. The flanking helix H of the metal binding site IV contains the residue 154. The absence of other Trp and Tyr residues in proteins enables unambiguous assignment of spectral properties.

Exposed alpha-helix links the two globular N and C domains of chicken troponin C (TnC). Escherichia coli exhibits the globular recombinant N and C domains which have F29 or W29 and F105 or W105 residues. The distortion of side-chain interaction among the D/E helix and several other residues is reduced by the presence of these domains which function as termination and initiation sites. The calcium ion stimulated structural transitions of N and C domains are monitored by W29 and W105 sites.

One- and two-dimensional NMR methods facilitate the analysis of a synthetic peptide antigen, representing the C-terminal domain of the Pseudomonas aeruginosa pilin strain K. The existence of the two beta-turns in the domain 134-142 is indicated by the ensemble of solution conformation, produced for PAK 128-144 trans. In solution, the antigenic peptides adopt a folded conformation.

Steady-state kinetic and single-turnover studies on the valine, serine, alanine and leucine L-lactate dehydrogenase of Bacillus stearothermophilus reveals that a threonine 246 hydroxyl group at active sites facilitates hydrolysis. The hydroxyl group is not important for substrate-binding to pyruvate. The mutation four dehydrogenases involves nucleotide thio analog-induced site-directed mutagenesis.

A caldesmon attraction column and spectroscopic methods such as circular dichroism and the fluorescent probe acrylodan are used to study the reaction between smooth muscle heavy meromyosin (HMM) and caldesmon. HMM binds to caldesmon with a stoichiometry and an attraction that are analyzed by a thiol-specific fluorescent probe. The reaction between caldesmon and myosin becomes feeble due to the occurrence of calcium, which exhibits the constitutions of caltropin.

The aqueous environment and lipid surfaces react due to the amphipathic alpha-helices of exchangeable apolipoproteins (apo). The absence of lipids leads to a self-association of apoA-I's in mammals, but the dissociation from a high-density lipoprotein (HDL) causes chicken apoA-I to become a monomeric protein. A midpoint of denaturation of 0.64 M guanidine hydrochloride is seen when lipid-free apoA-I is denatured and studied by circular dichroism (CS). Partial stability of lipid-free apoA-I can be obtained by forming intra-or inter-helix salt bridges between amino acid side chains, with opposite charges.

Lipid transfer protein (LTP) associated with lipid microemulsion is isolated from commercially available rabbit plasma to study the reactivity of LTP toward lipids and to analyze the protein structure. The process of its isolation involves the precipitation of lipoproteins from the plasma by dextran sulfate and their purification by column chromatographics through Butyl-Toyopearl 650M, CM-Toyopearl 650M and Butyl-Toyopearl 650 M. The process of sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in the migration of LTP as a single band with an anti-human LTP monoclonal antibody, TP2.

A study of an amphipathic alpha-helical model peptide showed each amino acid to posses a different helix propensity in an amphipathic alpha-helix, depending on its location in the hydrophobic or hydrophilic face. Experiments also analysed the effect of substitution on helix structure and stability, and found the effect of substitution to be more in the hydrophobic face. Peptide had a 65% alpha-helical structure in solution, with the helix formation being a mononuclear process.

The positional effects of the disulfide bond on the structure and stability of a two-stranded alpha-helical coiled-coil were investigated. An interchain disulfide bond was systematically introduced into the hydrophobic core of a de novo designed model at the N-terminus, C-terminus and nonterminal positions a and d. The results indicate that the most effective disulfide bonds are those that can be introduced in the hydrophoibic core without any disruption of the protein structure.