Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Ammonium induces a conformational change in the Dickerson dodecamer

Upon addition of ammonium chloride, the Dickerson dodecamer [d(CGCGAATTCGCG)]2 undergoes a limited but unambiguous conformational change which affects the AATT part of the duplex. The evidence consists primarily in a change in chemical shifts of A5(H2) and T7(H6) by respectively 0.3 and 0.05 ppm, and in a change in the nOe intensity of cross-peak A6(H2)-T8(H1') which translates into a change of the A6(H2)-T8(H1') distance from 0.407 nm to 0.348 nm (pH 8.8, 20¡C). The transition is specific of ammonium. It is complete at a concentration of 200 mM. It is not induced by high concentrations of sodium. Similar effects are observed in the center of the A-tract of [d(GCA4T4GC)]2, but not in [d(CGCGATCGCG)]2, suggesting that the conformational change is a feature of the DNA-curving structure of duplexes with A-tracts. The effect of ammonium may be related to its localization in the minor groove, which has been reported previously (1).

Further evidence for a conformational change is provided by the kinetics of base-pair opening as determined from imino proton exchange. At low ammonium concentration, the base-pair lifetime and dissociation constant of the A6.T pair are 74 ms and 5x10-6. At high ammonium concentration, they change to 19 ms and 1.7x10-6. These data are in agreement with an earlier report (2). It is noteworthy that in either condition, the time for ammonia-catalyzed exchange of the imino proton of base pair A6.T (and of others as well) is a linear function of the inverse of ammonia concentration, as expected for exchange from a single open state, provided that a constant concentration of ammonium (NH4+) is maintained while the ammonia (NH3 )concentration is varied.

In contrast, if ammonia and ammonium change concurrently, the exchange rate is affected by changes in both proton exchange catalysis and in conformation. In such conditions, a plot of the exchange time as a function of the inverse of ammonia concentration will be non-linear. This phenomenon was taken as evidence for proton exchange from a single native structure by way of two open states (2). The present observations show instead that the phenomenon has its origin in the occurence of two native conformations, and that for each of these, exchange of the imino proton with water proceeds via a single mode of base-pair opening.

References and Footnotes
  1. Hud, N.V., Sklenar, V. & Feigon, J. (1999) J.Mol.Biol. 286, 651-660.
  2. W?rml?nder, S., Sen, A. & Leijon, M. (2000) Biochemistry 39, 607-615.

K. Snoussi and J.-L. Leroy

Groupe de Biophysique du Laboratoire de Physique de la Mati?re Condens?e Ecole Polytechnique 91128 Palaiseau FRANCE
Tel.: 01 69 33 44 11; fax : 01 69 33 30 04; E.mail: ks@pmc.polytechnique.fr