Membrane proteins

Two protein tyrosine phosphatases, dual specificity phosphatase PVP and low molecular weight phosphatase WZB were purified and characterized. PVP was expressed as inclusion bodies and a suitable purification and refolding method was devised. Enzyme kinetics revealed that p-nitrophenylphosphate and (Sb(B-naphthyl phosphate were substrates with KM of 4.0mM and 8.1mM respectively. PVP showed no reactivity towards phosphoserine. Kinetic characterization of WZB showed that only pnitrophenylphosphate was a substrate with no affinity for Ç-naphthyl phosphate and phosphoserine. Optimal conditions for activity with PNPP were found at a pH of 5 with a KM of 1.1mM, kcat of 35.4s-1 and kcat/KM of 32.2s-1mM-1. Inhibition studies showed that phosphate, fluoride, and molybdate were competitive inhibitors with Ki of 3.2mM, 71.7mM, and 50.4(So(BM respectively and hydrogen peroxide abolished activity. Active site mutants of WZB Cys9Ser and Asp115Asn showed no activity.

Talin is a ubiquitous, high-molecular-weight, flexible protein that plays a critical role in focal adhesions by activating, as well as connecting, integrins to the actin cytoskeleton. Talin's inactive auto-inhibitory state is speculated to be one of its modes of regulation inside the cell and is achieved through its head-tail interactions. In order to decipher the stability of this interaction, the head domain (residues 206-405) was cloned into a modified pET28m vector while the tail domains (residues 1654-2344 and 2225-2344) were cloned into the pET32a vector to obtain octa-histidine tagged and un-tagged peptide, respectively. Neither co-expression nor pull-down using the His-tagged head domain was successful in purifying a stable head-tail complex. Our results indicate rather weak interactions between the talin head and rod domains and hence, under our experimental conditions, do not lead to a stable auto-inhibitory complex that can be purified for further studies.