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Results show that the activity restoration in the mutant thymidylate synthase follows a sequential pattern of subunit separation and their subsequent recombination to an active heterodimer. Sequence data of the homo- and heterodimers of the enzyme indicate that the active enzyme is a product of subunit exchange.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore ([[Lambda].sub.max] 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed [[Lambda].sub.max] at 500 nm, whereas that from L125A showed [[Lambda].sub.max] at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and [Delta]Y191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant [Delta]Y191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Differential scanning calorimetry helps examine the unfolding of barnase, an intermediate in the folding pathway of ribonuclease, at low pH to determine the A-state of barnase. A-state of barnase has high content of secondary structure and no fixed tertiary structure, and thermal denaturation leads to loss of cooperativity. Examination of various barnase mutants reveals the relation between conformational states and kinetic folding intermediate. Fluorescence spectroscopy helps analyze the thermal denaturation while kinetic and chemical cross-linking experiments suggest A-state as the main folding intermediate state of barnase.

Two amino acid substitutions at different regions of the P22 tailspike polypeptide chain of Salmonella typhimurium correct folding defects caused by mutations of the temperature sensitive tsf region. the suppressor mutations at codons 331 and 334 are crucial factors affecting tailspike folding. the substitution of an ionic pair of amino acids by a hydrophobic pair in the tsf revertant is a specific interaction caused by a suppressor.

The article the newly developed i-motif in the bcl-2 P1 promoter, which is shown to exhibit a stable structure with an 8:5:7 loop conformation. The loops presents in these motifs are shown to be highly applicable for the recognition of proteins and small molecules.

The article studies the crystal structures of lactose permease from Escherichia coli (LacY) that exhibits two six-helix bundles with 2-fold pseudosymmetry separated by a large hydrophilic cavity. Findings reveal that the primary defect induced by the mutation might include a decrease in affinity for [H.super.+].

The helical subdomain of the villin headpiece as the smallest naturally occurring cooperatively folded protein which have well-packed hydrophobic core comprised in part of an unusual set of three closely packed phenylalanine residues, F47, F51, and F58 is described. Small size, simple three-helix topology, and very rapid folding observed for the helical domain established it as an extremely popular model system for computational and theoretical studies of protein folding.

The effects of the mutants Y92G, Y92A, and Y92L on both the conformational and oxidative folding pathways were examined to determine the role of native interactions in different types of conformational searches for the biologically active structure of a protein, Ribonuclease A (RNase A). The results demonstrated the significance of native interactions in the folding process, namely, protection of a native (40-95) disulfide bond by a nearby tyrosyl-prolyl stacking interaction, when disulfide bonds are allowed to undergo SH/S-S reshuffling.

Several studies are conducted to determine the applicability of low-affinity zinc coordination motifs in mediating the destabilization of the close-dependent rhodopsin. The process is shown to be extremely useful for the prevention of various diseases, like retinitis pigmentosa (RP) and the Alzheimer disease.

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. 15 (Vib), is analyzed. The refolding processes of the mutants are followed by measuring changes in Trp fluorescence and the high similarity of time courses has suggested cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

A study is conducted to examine the deleterious mutants in the uroporphyrinogen III synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The data obtained from the integrated analysis of the enzymatic activity and kinetic stability offer insight into the relationship between defects in UROIIIS sequence and CEP.

The directed mutagenesis at 10 different interface contacts is carried out to gain insight into the role of intersubunit side chain-side chain interactions in the stability of the Escherichia coli aspartate aminotransferase (eAATase) homodimer. The cooperative unfolding of the dimeric intermediate (D*) is explained by the unfolding of the small domain that lead to monomer dissociation.

The transition state of ubiquitin thermal unfolding is probed by examining unfolding dynamics and kinetics of wild-type and mutant ubiquitin by using time-resolved nonlinear infrared spectroscopy after a nanosecond temperature jump. The increased and decreased downhill unfolding amplitudes of mutants i and g have supported the presence of an intermediate state on the native side of the rate-limiting barrier.

The tolerance of a natural thermostable chorismate mutase and an engineered molten globular variant to targeted mutation is compared to understand the mutational robustness of homologous enzymes that correlates with a higher initial free energy of unfolding ([DELTA]G). The mutases are found to have similar sequence, structure, and catalytic efficiency but different [DELTA]G values and the analogous point mutations could have widely divergent effects on catalytic activity in the scaffolds.

The dynamic structure and unfolding profiles of a dynein light chain LC8 mutant (H55K) are characterized as a model for monomeric LC8 at neutral pH. The thermodynamics of the LC8 binding to three peptides and the dynamics of the LC8-peptide complexes have shown a correlation between the flexibility of the binding groove and the entropic cost of binding and also that the change in flexibility of LC8 is ligand-dependent.

The protein modeling, chemical modification, and enzymatic assays were used to study the select exon 15-encoded N-terminal site-directed mutants and also determine the role of the putative conformational switch in arginine methylation. The data obtained supports a novel model for fragile X mental retardation protein (FMRP) arginine methylation in the RG-rich region and the structural changes in [beta]-sheet due to mutating R(507) in the conformation switch to K.

The design of a new model system based upon collagen-like heterotrimers that can mimic the glycine mutations present in either the [alpha]1 or [alpha]2 chains of type I collagen is reported. The results obtained demonstrate the difference in thermal stability and refolding half-life times between triple helices that vary only in the frequency of glycine mutations at a particular position.

The impact of the expansion of the octarepeat (OR) domain on the prion-like conformation of the prion protein (PrP) is discussed. The expansion is shown to affect the misfolding pathway of PrP, where as the folding pathway remains unaffected.

An investigation of the dynamics of helix C in the free U1A protein is reported. The results show that helix C is dynamic on a 2-3 nanoseconds time scale and its dynamical motion occurs within the closed conformation.

The Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation dispersion study for the monomer-dimer equilibrium of the GB1 point mutant, Ala-34-Phe ([GB1.sup.A34F]) is reported. The dispersion experiment is useful for studying association equilibria due to its sensitivity to minor population in the equilibrium.

The thermodynamic properties of the C- domain of TnC in its apo, [Mg.sub.2+] and [Ca.sub.2+] states are studied using a fluorescent mutant. The N-domain of TnC did not seem to have any effect on the C- domain, while a blue shift was observed in the Trp emission on binding with [Mg.sub.2+] and [Ca.sub.2+].

The studies were conducted to determine the structure, functions and biosynthetic processes related to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The results obtained provide insight into CFTR epithelial anion channel which causes cystic fibrosis (CF).

Research points out that in the villin headpiece, cycles of scaffolding patterns induce helix formation at very low local propensity. Furthermore, site mutations in villin protein lead to marked changes in the kinetics of protein folding.