Measurement of ligand binding to tubulin by sulfhydryl reactivity.
Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule polymerization. 5',5'-Dithiobis(2-nitrobenzoate) (DTNB) reacts with free and accessible sulfhydryls and stoichiometrically produces a detectable product, which allows an exact measurement of reacted thiols. Since binding of small ligands may alter conformational dynamics, it may also affect the reactivity of thiols on tubulin. Differences in DTNB reactivity with thiols upon ligand binding can therefore be used to deduce binding characteristics. We will describe two methods that use tubulin cysteine reactivity with DTNB in the presence of drug to define ligand-binding characteristics.
Begaye
Adrian
A
Laboratory of Integrative and Medical Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Sackett
Dan L
DL
D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000494:pharmacology*; D014404:Tubulin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D050257:Tubulin Modulators / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D014404:Tubulin; D050257:Tubulin Modulators
Anal Biochem. 2003 Jan 15;312(2):224-7
12531209
Anal Biochem. 2004 Sep 15;332(2):376-83
15325307
Methods Mol Med. 2007;137:281-8
18085236
Biochem J. 2005 Jul 15;389(Pt 2):549-58
15743274
Curr Med Chem Anticancer Agents. 2002 Jan;2(1):71-90
12678752
Pharmacol Ther. 1993 Aug;59(2):163-228
8278462
J Mol Biol. 2008 May 16;378(5):1016-30
18405918
J Biol Chem. 1977 Feb 25;252(4):1134-40
14143
Bioorg Med Chem. 2008 Jun 1;16(11):6269-85
18468444
Pharmacol Ther. 1991;49(1-2):133-52
1852786
Eur J Biochem. 2000 Jun;267(12):3469-76
10848962
J Biol Chem. 2002 Aug 9;277(32):29018-27
12023292
Annu Rev Biochem. 2006;75:519-41
16756501
Methods Mol Med. 2007;137:17-28
18085219