Exo-mechanism proximity-accelerated alkylations: investigations of linkers, electrophiles and surface mutations in engineered cyclophilin-cyclosporin systems.
Investigations on the scope and utility of exo-mechanism proximity-accelerated reactions in engineered receptor-ligand systems are reported. We synthesized a series of electrophilic cyclosporin (CsA) derivatives by varying electrophiles and linker lengths, prepared a series of nucleophilic cysteine mutations on the surface of cyclophilin A (Cyp), and examined their reactivity and specificity in proximity-accelerated reactions. Acrylamide and epoxide electrophiles afforded useful reactivity and high specificity for alkylation of engineered receptors in Jurkat cell extracts. We found that remote cysteines (>17 A from the ligand) could be alkylated with useful rates under physiological conditions. The results from mutations of the receptor surface suggest that the dominant factors governing the rates of proximity-accelerated reactions are related to the local environment of the reactive group on the protein surface. This study defines several parameters affecting reactivity in exo-mechanism proximity-accelerated reactions and provides guidance for the design of experiments for biological investigations involving proximity-accelerated reactions.
Levitsky
Konstantin
K
Department of Biochemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.
Boersma
Melissa D
MD
Ciolli
Christopher J
CJ
Belshaw
Peter J
PJ
D000478:Alkylation; D003432:Cross-Linking Reagents; D021983:Cyclophilins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003524:Cyclosporins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D003524:Cyclosporins; D021983:Cyclophilins; D003545:Cysteine