Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2'(3')-O-(-N-Methylanthraniloyl)-adenosine 5'-triphosphate.
MantATP [2'(3')-O-(-N-methylanthraniloyl)-adenosine 5'-triphosphate] was employed as a fluorescence probe of the nucleotide-binding sites of dynein from sea urchin sperm flagella. MantATP binds specifically with enhanced fluorescence (approximately 2.2-fold), homogeneous lifetime (8.4 ns), and high anisotropy (r approximately 0.38) to dynein and can be displaced by ATP and ADP added to the medium. The association constants of mantATP complexed with dynein were determined from anisotropy titration data. Using a multiple stepwise equilibrium model, the average values of the first two association constants are K1 = 2.7 x 10(5) M-1 and K2 = 1.8 x 10(4) M-1. This value of K1 is 7-8 times higher than that found previously for unsubstituted ATP, whereas K2 is little changed [Mocz and Gibbons (1996) Biochemistry 35, 9204-9211]. The lower-affinity binding sites, K3 and K4, observed previously could not be studied with mantATP within the available protein concentrations. The alpha and beta heavy chain subfractions have binding parameters similar to those of intact dynein. Formation of the stable ternary complex of mantATP with dynein and monomeric vanadate is accompanied by only a moderate increase in the binding affinities. Oligomeric vanadate reduces the binding affinities by approximately 50%. Addition of TritonX-100, methanol, or various salts changes the binding affinities by up to 50%, suggesting that the microenvironment of the nucleotide-binding sites involves significant contributions from both polar and apolar interactions. The distinct affinities of the individual binding sites are consistent with a physiological role in regulating nucleotide binding.
Mocz
G
G
Pacific Biomedical Research Center, University of Hawaii at Manoa, Honolulu 96822, USA. gmocz@hawaii.edu
Helms
M K
MK
Jameson
D M
DM
Gibbons
I R
IR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism; D005407:Flagella / Q000201:enzymology; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D012492:Salts; D012617:Sea Urchins; D012997:Solvents; D013094:Spermatozoa / Q000201:enzymology; D062367:ortho-Aminobenzoates / Q000737:chemistry* / Q000378:metabolism
D000345:Affinity Labels; D005456:Fluorescent Dyes; D012492:Salts; D012997:Solvents; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D004398:Dyneins