Determination of the radial coordinate of Cys-374 in F-actin using fluorescence resonance energy transfer spectroscopy: effect of phalloidin on polymer assembly.
In helically symmetric protein assemblies, fluorescence resonance energy transfer (FRET) spectroscopy can be used to determine the radial coordinates of fluorescent probes attached to specific amino acid side chains. This is done by separately labeling monomers with donor and acceptor probes, mixing them in different proportions, allowing the mixtures to self-assemble, and then measuring the fluorescence intensity. Provided the helical symmetry is known, and provided the donor- and acceptor-labeled monomers associate randomly, the radial coordinate of the probes can be calculated. Using different probe pairs, two different research groups have employed this method to show that the Cys-374 site of the actin filament (F-actin) is located at a radius of either 35-40 A [Taylor, D.L., Reidler, J., Spudich, J.A., & Stryer, L. (1981) J. Cell Biol. 89, 362-367] or 20-25 A [Kasprzak, A.A., Takashi, R., & Morales, M.F. (1988) Biochemistry 27, 4512-4522]. We have reinvestigated these disparate radius determinations using the same probe pairs employed by these authors with a wide range of acceptor molar fractions. Our results suggest that labeling actin with probes makes the association of monomers significantly nonrandom. This may be avoided by polymerizing actin in the presence of phalloidin. The nonrandomness also can be modeled using stochastic simulation. Taking the average diameters of the probes into account, we conclude that in phalloidin-stabilized F-actin, Cys-374 lies at a radius of (17-18) +/- 5 A. This value is consistent with radial coordinates determined by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
Moens
P D
PD
Department of Anatomy and Histology, University of Sydney, Australia.
Yee
D J
DJ
dos Remedios
C G
CG
D000199:Actins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes; D008956:Models, Chemical; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000737:chemistry; D010590:Phalloidine / Q000737:chemistry*; D011108:Polymers; D011817:Rabbits; D012015:Reference Standards; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry
D000199:Actins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011108:Polymers; D013439:Sulfhydryl Reagents; D010590:Phalloidine; C015296:5-iodoacetamidofluorescein; C006421:1,5-I-AEDANS; D003545:Cysteine