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Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the water-filled cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure.

Research has been conducted on the hydrophobic core of ribonuclease T1. The use of the differential scanning calorimetry, urea denaturation and X-ray crystallography in investigating the structural and energetic results of the hydrophobic core's engineered cavity refilling with CH (sub)3, SH and OH groups is described.

Research has been conducted on the buried and well-ordered solvent molecules. The exchange kinetics of the water molecules with solvent has been examined.

Hydrogen-exchange rates for RNase T1 and variants with Ala results in Gly substitutions at a solvent-exposed position in the helix have been measured. Results demonstrate the change of the hydrogen-exchange stabilities of residues by Ala results in Gly mutations.

Phosphodiester transesterification process involves the Glu58 which acts as a catalyst along with the RNase T1 enzyme. The Glu58 RNase T1 participates in the process by an altered mechanism where the Glu58 is replaced by His40 as a base catalyst. Guanine 2'-monophosphate is cocrystallized with Glu58Ala RNase T1 during the process. The structure of the Glu58 is obtained by the nominal resolution method.

The X-ray structure, gene structure and amino-acid arrangement of the orthorhombic crystal of the lentil lectin at 2.3-angstrom resolution are examined. Crystallography is used to study the high-resolution legume lectin structure. The geometrics of pea and lathyrus lectin are contrasted with that of the crystal. The binding of the carbohydrates and the activation of the posttranslation are affected by the structure. Hundred-and-eighty amino acids are included in the amino acid arrangement of the beta-chain. The temporary metal-binding site shows distinct variations in structure.

A study was conducted on the participation of histidine-40 in ribonuclease T1 catalysis. Three-dimensional crytsal structures of the His40Lys mutant were analyzed. The results revealed that His40Lys mutation does not induce major changes in the structure of RNase T1. Further observations also showed that Lys40 side chains existing in the mutant structures inhabit almost the same space.

The first functional and structural characterization of the inosine-guanosine-specific nucleoside hydrolase (NH) from Trypanosoma brucei brucei is reported. The study identified nanomolar iminoribitol-based inhibitors which could be important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.

The transient kinetics of the nucleoside hydrolase from Trypanosoma vivax (TvNH) at low temperatures (5 degree Celsius) is investigated. Together with steady-rate product inhibition results, the data indicate that TvNH follows an ordered uni-bi kinetic mechanism with a TvNH-base dead-end complex, and not the rapid equilibrium random uni-bi mechanism proposed for other NHs.

The functional characterization of the Escherichia coli YbeK (RihA) protein as a pyrimidine nucleoside-preferring nucleoside hydrolases (NH), and its first crystal structure to 1.8 Angstrom resolution is presented. The enzyme active site by alpha or beta anomer of ribose and provides the first structural description of the binding of the NH reaction product, while the amino acid residues involved in ribosyl binding are conserved in pyrimidine specific NHs.

Results demonstrate that nucleoside hydrolase of Trypanosoma vivax displays burst kinetics with half-of-the-sites reactivity. The enzyme exhibits a complex multistep mechanism with a rate limiting slow isomerization between a tight and a loose enzyme-ribose complex, which accumulates during the enzyme hydrolysis of purine nucleosides.

Presents the crystal structure of Glu58Ala RNase T1 complexed with the specific inhibitor 2'-GMP in space group P2(1). Structural aspects of the change in enzyme mechanism associated with the Glu58Ala mutation; Implications of the difference in space group on the structure of the protein and its hydration shell.