Charge-Shielding and the “Paradoxical” Stimulation of Tubulin Polymerization by Guanidine Hydrochloride

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Abstract
Low concentrations of guanidine hydrochloride (GuHCl) increase the rate (and to a lesser degree, the extent) of tubulin polymerization as assessed by light scattering. Maximum enhancement occurs at 120−160 mM GuHCl followed by decreases at higher GuHCl. The latent period is decreased, and there is a 3−4-fold reduction in the critical concentration for polymerization. Electronmicrographs reveal microtubules in the controls and an increasing fraction of total polymers present as aberrant microtubules as the GuHCl concentration is increased from 20 to 100 mM. The GuHCl effect is markedly reduced, but not abolished, in tubulin S (in which the anionic C termini of both monomers have been removed). The GuHCl-induced polymerization has an absolute requirement for GTP and taxol or DMSO, is very sensitive to podophyllotoxin inhibition, and can overcome urea-mediated inhibition of polymerization. Guanidinium analogues mimic the GuHCl effect roughly as a function of the number of potential hydrogen bonds. The anions of the guanidine salts superimpose their inhibitory action on the guanidinium cation effect according to the lyotropic series. At higher GuHCl concentrations (peak effect 500−700 mM), a different polymer (type II) is formed that is GTP and taxol independent, but whose polymerization is retarded but not prevented by podophyllotoxin. Its structure resembles the fibrillar network seen in unfolding intermediates of other proteins. We conclude that both charge and hydrogen-bonding ability are major contributors to the GuHCl-induced promotion of tubulin polymerization, and that charge-shielding is likely to be the basis for this effect.

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