Fluorophore-assisted light inactivation of calmodulin involves singlet-oxygen mediated cross-linking and methionine oxidation.
Fluorophore-assisted light inactivation (FALI) permits the targeted inactivation of tagged proteins and, when used with cell-permeable multiuse affinity probes (MAPs), offers important advantages in identifying physiological function, because targeted protein inactivation is possible with spatial and temporal control. However, reliable applications of FALI, also known as chromophore-assisted light inactivation (CALI) with fluorescein derivatives, have been limited by lack of mechanistic information regarding target protein sensitivity. To permit the rational inactivation of targeted proteins, we have identified the oxidizing species and the susceptibility of specific amino acids to modification using the calcium regulatory protein calmodulin (CaM) that, like many essential proteins, regulates signal transduction through the reversible association with a large number of target proteins. Following the covalent and rigid attachment of 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) to helix A, we have identified light-dependent oxidative modifications of endogenous methionines to their corresponding methionine sulfoxides. Initial rates of methionine oxidation correlate with surface accessibility and are insensitive to the distance between the bound fluorophore and individual methionines, which vary between approximately 7 and 40 A. In addition, we observed a loss of histidines, as well as zero-length cross-linking with binding partners corresponding to the CaM-binding sites of smooth myosin light chain kinase and ryanodine receptor. Our results provide a rationale for proteomic screens using FALI to inhibit the function of many signaling proteins, which, like CaM, commonly present methionines at binding interfaces.
Yan
Ping
P
Cell Biology and Biochemistry Group, Division of Biological Sciences, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Xiong
Yijia
Y
Chen
Baowei
B
Negash
Sewite
S
Squier
Thomas C
TC
Mayer
M Uljana
MU
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008027:Light; D013058:Mass Spectrometry; D008715:Methionine / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D026082:Singlet Oxygen / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D002147:Calmodulin; D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D010446:Peptide Fragments; D010455:Peptides; D017382:Reactive Oxygen Species; D019837:Ryanodine Receptor Calcium Release Channel; C090954:SM 1 peptide; D026082:Singlet Oxygen; D006639:Histidine; D008715:Methionine; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin; D003545:Cysteine