02464nas a2200217   4500008004100000022001400041245013200055210006900187260007500256300001400331490000700345520161200352653002701964653002201991653001702013653002002030653001502050100002502065700002502090856013102115       2014                        eng d  a1053-050900aTryptophan environment and functional characterization of a kinetically stable serine protease containing a polyproline II fold0 aTryptophan environment and functional characterization of a kine  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScSEP  a1363-13700 v243 a<p>The single tryptophan residue from Nocardiopsis sp. serine protease (NprotI) was studied for its microenvironment using steady state and time-resolved fluorescence. The emission maximum was observed at 353 nm with excitation at 295 nm indicating tryptophan to be solvent exposed. Upon denaturation with 6 M guanidinum thiocyanate (GuSCN) the emission maxima was shifted to 360 nm. Solute quenching studies were performed with neutral (acrylamide) and ionic (I- and Cs+) quenchers to probe the exposure and accessibility of tryptophan residue of the protein. Maximum quenching was observed with acrylamide. In the native state, quenching was not observed with Cs+ indicating presence of only positively charged environment surrounding tryptophan. However; in denatured protein, quenching was observed with Cs+, indicating charge reorientation after denaturation. No quenching was observed with Cs+ even at pH 1.0 or 10.0; while at acidic pH, a higher rate of quenching was observed with KI. This indicated presence of more positive charge surrounding tryptophan at acidic pH. In time resolved fluorescence measurements, the fluorescence decay curves could be best fitted to monoexponential pattern with lifetimes of 5.13 ns for NprotI indicating one conformer of the trp. Chemical modification studies with phenyl glyoxal suggested presence of Arg near the active site of the enzyme. No inhibition was seen with soyabean trypsin and limabean inhibitors, while, CanPI uncompetitively inhibited NprotI. Various salts from Hofmeister series were shown to decrease the activity and PPII content of NprotI.</p>10aFluorescence quenching10aHofmeister series10aNocardiopsis10aSerine protease10aTryptophan1 aRohamare, Sonali, B.1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/tryptophan-environment-and-functional-characterization-kinetically-stable-serine-protease-0