No description available.

It is critical to consider the balance between the catalytic capabilities of an enzyme and the inherent structural stability of the protein when developing enzymes for specific applications. Rational site directed mutagenesis has been used to explore the role of residues 254 and 257 in the global stability and catalytic specificities of organophosphorus hydrolase (OPH, EC 3.1.8.1). Substitution of residues H254 and H257, which are located near the active site, had a marked effect on both the global stability and substrate specificity of the enzyme. For example, the for the double mutation CoTGΔ2+ H254R H257L (RL) enzyme variant was 19.6 kcal/mol, 5.7 kcal/mol less than that of the wild type enzyme. At the same time, the altered enzyme was catalytically more effective against VX and VR (Russian VX), as compared to the wild type enzyme. Limited proteolysis verified the importance of residues 254 and 257 for functional stability, evidenced by enhanced resistance to irreversible unfolding associated with thermal denaturation. It has been possible to construct third generation OPH variants, which are more stable than the wild type enzyme, with a 10 °C increase in the apparent melting temperature (TM app), yet retained desirable catalytic properties. It appeared that aromatic stacking and cation-π interactions involving near active site residues not only affected activity but significantly contributed to the chemical and thermal stability of OPH. Rational design was used to develop an enzyme with an optimized orientation on a catalytically active biosensor surface. In these studies, lysine side chains located on the surface of OPH were used to create attachment sites to a surface plasmon resonance sensor resulting in an ensemble of enzyme orientations. Some of these orientations could be functionally restrictive if the active site is oriented toward the sensor surface. Substitution of a lysine near the active site resulted in 20% more activity with 53% less enzyme immobilized, thus increasing the specific activity of the decorated surface 2.5 fold.

It is critical to consider the balance between the catalytic capabilities of an enzyme and the inherent structural stability of the protein when developing enzymes for specific applications. Rational site directed mutagenesis has been used to explore the role of residues 254 and 257 in the global stability and catalytic specificities of organophosphorus hydrolase (OPH, EC 3.1.8.1). Substitution of residues H254 and H257, which are located near the active site, had a marked effect on both the global stability and substrate specificity of the enzyme. For example, the for the double mutation CoTGΔ2+ H254R H257L (RL) enzyme variant was 19.6 kcal/mol, 5.7 kcal/mol less than that of the wild type enzyme. At the same time, the altered enzyme was catalytically more effective against VX and VR (Russian VX), as compared to the wild type enzyme. Limited proteolysis verified the importance of residues 254 and 257 for functional stability, evidenced by enhanced resistance to irreversible unfolding associated with thermal denaturation. It has been possible to construct third generation OPH variants, which are more stable than the wild type enzyme, with a 10 °C increase in the apparent melting temperature (TM app), yet retained desirable catalytic properties. It appeared that aromatic stacking and cation-π interactions involving near active site residues not only affected activity but significantly contributed to the chemical and thermal stability of OPH. Rational design was used to develop an enzyme with an optimized orientation on a catalytically active biosensor surface. In these studies, lysine side chains located on the surface of OPH were used to create attachment sites to a surface plasmon resonance sensor resulting in an ensemble of enzyme orientations. Some of these orientations could be functionally restrictive if the active site is oriented toward the sensor surface. Substitution of a lysine near the active site resulted in 20% more activity with 53% less enzyme immobilized, thus increasing the specific activity of the decorated surface 2.5 fold.

No description available.

Abstract¡G Tumor susceptibility gene 101, TSG101, is a protein exhibits multiple biological functions. For most protein, its specific function or structure stability can be regulated through protein phosphorylation or modification. The analysis of the amino acid sequence of TSG101 revealed that it has two GSK-3£] phosphorylation concensus sequences. Our previous data of in vitro kinase assay have demonstrated that TSG101 can be phosphorylated by GSK-3£], a wellknown protein kinase that regulates the stability and function of it¡¦s target protein. To investigate the effect of GSK-3£] phosphorylation on the stability and the function of TSG101 protein, we first exploited the effect of GSK-3£]inhibitor, LiCl, on endogenous TSG101 protein in COS1 cells. The results suggested that inhibition of GSK-3£] phosphorylation could impact on the stability of TSG101 protein. Upon the transfection of an active form GSK-3£] expression plasmid GSK-3£]/pEGFP, additional protein products of 40, 50-80 kD were detected, suggesting that GSK-3£] phosphorylation might induce modification or degradation of TSG101 protein. GSK-3£] phosphorylation site mutant TSG101 protein expression plasmids were constructed using site-directed mutagenesis, and were transfected into COS1 cells to evaluate the effect of GSK-3£] on TSG101 level. The results showed that GSK-3£] phosphorylation site mutant TSG101 protein is more stable then wild type TSG101 due to the lack of GSK-3£] phosphorylation site. The inhibition of GSK-3£] activity by LiCl treatment resulted in the increase of wildtype as well as the S172AS176 and S172AS176AS202AS206A mutant TSG101 proteins, whereas the S202AS206A mutant TSG101 protein level was not affected by LiCl treatment. The above data indicated that GSK-3£] might regulate the stability and biological activity of TSG101 protein through phosphorylation of serine residue at position 202, which is worthy of further investigation.

Proteins need fold to perform their biological function. Thus, understanding how proteins fold could be the key to understanding life. In the first study, the stability and structure of several !-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G (PGB1) were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the !-turn conformation. Our results indicate that the structure and stability of this !?hairpin peptide can be modulated in numerous ways and thus contributes towards a more complete understanding of this important model !-hairpin as well as to the folding and stability of larger peptides and proteins. The second study revealed that PGB1 and its variants can form amyloid fibrils in vitro under certain conditions and these fibrils resemble those from other proteins that have been implicated in diseases. To gain a further understanding of molecular mechanism of PGB1 amyloid formation, we designed a set of variants with mutations that change the local secondary structure propensity in PGB1, but have similar global conformational stability. The kinetics of amyloid formation of all these variants have been studied and compared. Our results show that different locations of even a single mutation can have a dramatic effect on PGB1 amyloid formation, which is in sharp contrast with a previous report. Our results also suggest that the "-helix in PGB1 plays an important role in the amyloid formation process of PGB1. In the final study, we investigate the forces that contribute to protein stability in a very general manner. Based on what we have learned about the major forces that contribute to the stability of globular proteins, protein stability should increase as the size of the protein increases. This is not observed: the conformational stability of globular proteins is independent of protein size. In an effort to understand why large proteins are not more stable than small proteins, twenty single-domain globular proteins ranging in size from 35 to 470 residues have been analyzed. Our study shows that nature buries more charged groups and more non-hydrogen-bonded polar groups to destabilize large proteins.

The Large Tumor Suppressor 1 (LATS1) is a serine/threonine kinase and tumor suppressor found down-regulated in a broad spectrum of human cancers. It is a central player of the emerging Hippo-LATS tumour suppressor pathway, which plays important roles in cell proliferation, apoptosis, and stem cell differentiation. Despite the ample data supporting a role of LATS1 in tumour suppression, how LATS1 is regulated at the molecular level remains largely unknown. In this study, we have identified Itch, a HECT class E3 ubiquitin ligase, as a novel binding partner of LATS1. Itch can complex with LATS1 both in vitro and in vivo through the PPxY motifs of LATS1 and the WW domains of Itch. Significantly, we found that over-expression of Itch promoted LATS1 degradation by polyubiquitination through the 26S proteasome pathway. On the other hand, knockdown of endogenous Itch by shRNAs provoked stabilization of endogenous LATS1 proteins. Finally, through several functional assays, we also revealed that change of Itch abundance alone is sufficient for altering LATS1-mediated downstream signaling, negative regulation of cell proliferation, and induction of apoptosis. Together, our study identifies E3 ubiquitin ligase Itch as the first negative regulator of LATS1 and presents for the first time a possibility of targeting LATS1/Itch interaction as a therapeutic strategy in cancer. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2011-04-26 22:25:46.008

Alzheimer’s disease (AD) patients could benefit from a more effective treatment than the current FDA-approved options. Because amyloid-beta (Aβ) is thought to play a central role in AD pathogenesis, many experimental drugs attempt to reduce Aβ-induced pathology. Preventing amyloid accumulation may be a more effective strategy than clearing Aβ plaques after they form. If preventing Aβ accumulation can treat or prevent AD, then understanding Aβ primary nucleation may aid rational drug design. This study examines Aβ residue interaction networks and reports network and structural observations that may provide insight into primary nucleation. While many studies identify structural features of Aβ that promote aggregation, this study reports features that may resist primary nucleation by examining Aβ42 studies in more and less polar solvents. In Aβ42 in a less polar solvent (PDB ID: 1IYT), Val24 and Ile31 have higher betweenness and residue centrality values. This may be due to a predicted interaction between Val24 and Ile31. Residues in the central hydrophobic cluster (CHC) of Aβ40 and Aβ42 had significantly higher betweenness values compared to the average betweenness of the structures, highlighting the CHC’s reported role in oligomerization. The predicted interaction between Val24 and Ile31 may reduce the likelihood of primary nucleation of Aβ.

No description available.

No description available.