Effects of fluorescent probe structure on the dynamics at cysteine-34 within bovine serum albumin: evidence for probe-dependent modulation of the cybotactic region.
We have prepared a series of bovine serum albumins (BSA) that have been site-selectively labeled at cysteine-34 with one of four different sulfhydryl-selective boron dipyrromethene difluoride (BODIPY) fluorescent probes (BODIPY FL IA, BODIPY FL C(1) IA, BODIPY 530/550 IA, and BODIPY 493/503 MB). We determine how the choice of extrinsic probe structure dictates the recovered BSA-BODIPY dynamics under thermal (10-80 degrees C) and chemical (0-5M guanidine hydrochloride) denaturation conditions. The results of these experiments show that the global protein dynamics are sensed equally by each fluorescent probe; however, the probe itself influences the local probe dynamics within the cybotactic region that surrounds cysteine-34. Thus, it seems inappropriate to think of these extrinsic fluorescent probes as passive, nonparticipatory viewers of local protein dynamics.
Baker
G A
GA
Department of Chemistry, Natural Sciences Complex, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA.
Pandey
S
S
Kane
M A
MA
Maloney
T D
TD
Hartnett
A M
AM
Bright
F V
FV
D000818:Animals; D001896:Boron Compounds; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000378:metabolism*; D006358:Hot Temperature; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation / Q000502:physiology; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine; D019791:Guanidine; D003545:Cysteine