02592nas a2200277   4500008004100000022001400041245008800055210006900143260007300212300001200285490000900297520161200306653001701918653003801935653002201973653002101995653002102016653002002037100002502057700002002082700002602102700002402128700002102152700002502173856011602198       2013                        eng d  a1570-963900aPolyproline fold-In imparting kinetic stability to an alkaline serine endopeptidase0 aPolyproline foldIn imparting kinetic stability to an alkaline se  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAR  a708-7160 v18343 a<p>Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsis sp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme in presence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functional activity and conformational transitions of the enzyme were studied under various denaturing conditions. Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0 M concentration, organic solvents viz, methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymottypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with a high energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzyme was observed in 1 M GdnHCl upto 8 h, in organic solvents (75% v/v) for 72 h and in presence of proteolytic enzymes. The polyproline fold remained unaltered or became more prominent under the above mentioned conditions. However, it diminished gradually during thermal denaturation above 60 degrees C. Thermal transition studies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibility of denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPII helix being the global conformation of a non structural protein, an alkaline serine protease, from a microbial source, imparting kinetic stability to the protein. (C) 2013 Elsevier B.V. All rights reserved.</p>10aConformation10adifferential scanning calorimetry10aKinetic stability10aNocardiopsis sp.10aPolyproline fold10aSerine protease1 aRohamare, Sonali, B.1 aDixit, Vaishali1 aNareddy, Pavan, Kumar1 aSivaramakrishna, D.1 aSwamy, Musti, J.1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/polyproline-fold-imparting-kinetic-stability-alkaline-serine-endopeptidase-002464nas 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