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MutL is required to assist the mismatch repair protein MutS during initiation of the methyl-directed mismatch repair (MMR) response in various organisms ranging from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity of MutL to aggregate has led to significant difficulties in determining its biological relationship with other MMR-related proteins. Here, we perform analysis on the thermostable MutL protein found in Thermotoga maritima MSB8 (TmL). Size exclusion chromatographic analysis indicates the lack of aggregated forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS) analysis reveals that the solution structures of the full-length TmL and its corresponding complexes with nucleotides and ssDNA undergo conformational changes. The elucidated TmL SAXS model is superimposed to the crystal structure of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal SAXS model of TmL exists as monomeric form, indicating that TmL has a structurally flexible N-terminal domain. TmL SAXS analysis can suggest a considerable possibility on a new 3D view of the previously unresolved full-length MutL molecule. [ABSTRACT FROM PUBLISHER]

One of important issues in protein science is to increase the protein stability. Utilizing a ketosteroid isomerase (KSI) as a model system, we constructed a mutant KSI which has a higher stability than its WT. We focused on the role of aromatic moieties of three tyrosine residues, Tyr14, Tyr55, and Tyr30, which constitute a hydrogen bond network in KSI. Therefore, we made a series of mutants which could substitute tyrosine with phenylalanine to remove the hydroxyl group in the tyrosine residue. We measured the free-energy change for unfolding using urea as a denaturant. We investigated the interaction among these aromatic residues for stability using the double mutant cycle analysis. We found that additional mutations on Y14F, Y55F, and Y14F/Y55F, respectively, by replacement of tyrosine at the position 30 with phenylalanine increased the stability ca. 4.0 kcal/mol. Especially the triple mutant KSI (Y14F/Y30F/Y55F) was more stable 2 kcal/mol than wild type KSI. Our structural analyses suggests that the enhanced stability of the triple mutant should be due to increased both hydrophobic interaction and aromatic-aromatic interaction upon the mutation [ABSTRACT FROM AUTHOR]

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-15N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the β5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the β6-strand. The results thus suggest that the interface region primarily around the β5- and β6-strands could play an important role as the starting positions in the unfolding process of KSI. [ABSTRACT FROM PUBLISHER]

The article informs about research on discovery of direct-acting antiviral agents with a graphene-based fluorescent nanosensor. Topics discussed include direct-acting drugs exist for the treatment against dengue virus (DV) infection, which can develop into life-threatening diseases; and development of an RNA-based graphene biosensor system [RNA nano-graphene oxide system (RANGO)] to enable the fluorescence-based quantitative analysis of the RdRp enzyme activity.

The backbone dynamics of Y14F mutant of Δ5-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS. [ABSTRACT FROM PUBLISHER]