title: Structural-functional insights of single and multi-domain Capsicum annuum protease inhibitors.
abstract: Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural-functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K(i)~57 μM) than -15 (~184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.
authors.affiliation: Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008, MS, India.
authors.forename: Manasi
authors.initials: M
authors.lastname: Mishra
authors.forename: Rakesh S
authors.initials: RS
authors.lastname: Joshi
authors.forename: Sushama
authors.initials: S
authors.lastname: Gaikwad
authors.forename: Vidya S
authors.initials: VS
authors.lastname: Gupta
authors.forename: Ashok P
authors.initials: AP
authors.lastname: Giri
mesh_terms: D000818:Animals; D002212:Capsicum / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C039304:calpain inhibitors; D014357:Trypsin