Evaluating electrostatic contributions to binding with the use of protein charge ladders

Science

Volumn 272, Issue 5261, 1996, Pages 535-537

  Gao, Jinming ^a   Mammen, Mathai ^a   Whitesides, George M ^a  

^a HARVARD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CARBONATE DEHYDRATASE II;

ACETYLATION; ARTICLE; BINDING AFFINITY; CAPILLARY ELECTROPHORESIS; CATTLE; ELECTRICITY; ENERGY; LIGAND BINDING; MOLECULAR RECOGNITION; NONHUMAN; PRIORITY JOURNAL; PROTEIN BINDING;

EID: 0029984673     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5261.535     Document Type: Article

References (21)

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4. The observation that interactions between colloidal particles of like charge can, in certain instances, be attractive further complicates our understanding of electrostatic effects in ionic media [N. Ise and H. Yoshida, Acc. Chem. Res. 29, 3 (1996), K S Schmitz, ibid., p 7].
5. The observation that interactions between colloidal particles of like charge can, in certain instances, be attractive further complicates our understanding of electrostatic effects in ionic media [N. Ise and H. Yoshida, Acc. Chem. Res. 29, 3 (1996), K S Schmitz, ibid., p 7].
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8. S. J. Dodgson, R. E. Tashian, G Gros, N. D Carter, Eds , The Carbonic Anhydrases. Cellular Physiology and Molecular Genetics (Plenum, New York, 1991).
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11. H. A. Abramson, L. S Moyer, M H. Gorin, Electrophoresis of Proteins and the Chemistry of Cell Surfaces (Reinhold, New York, 1942), chaps. 5 and 6
12. The charge ladder of CAII was prepared as follows The pH of three solutions of native CAII (0.1 mM in distilled water, 0.5 ml) was adjusted to pH 10 by addition of 0.1 N NaOH and was maintained at this value Acetic anhydride (10, 20, and 40 equiv, respectively, 100 mM in dioxane) was added After 30 mm, the three solutions were mixed and diluted to 10 μM in a buffer of 25 mM tris and 192 mM Gly (pH = 8 3) This procedure gave a sample in which each member of the charge ladder could be easily analyzed The neutral marker was 4-methoxybenzyl alcohol (50 μM).
13. We used a Beckman P/ACE 5500 system for CE General conditions:; operated at 15 kV, temperature T = 37°C, maintained by liquid cooling; capillary inner diameter, 50 μm; total length of capillary, 47 cm; length from inlet to detector, 40 cm. For the ACE experiments, different concentrations of a ligand were prepared in a buffer of 25 mM tris and 192 mM Gly (pH = 8 3). The capillary was flushed with 0.1 N NaOH, distilled water, and electrophoresis buffer for 2 min before each experiment
14. Using competitive ACE (8), we measured the binding affinities of the neutral ligands 2 and 3 to CAII by competition with ligand 1 (+1 charge), and that of neutral ligand 7 by competition with ligand 5 (-1 charge).
15. b.
16. a of sutfonamide groups or hydrophobic interactions) must contribute to differences in binding
17. The dimensions of CAII taken from its crystal structure are 41 Å by 41 Å by 47 Å [A. Liljas et al , Nature 235, 131 (1972)].
18. o = -35) at pH 8 3 to the fractional protonation of charged residues such as His or water bound to Zn(II)
19. Dissolved salts should lower this calculated electrostatic cost further by partially screening the charges on CAII from those on ligands
20. -1 In all three systems, opposite charges stabilized the receptor-ligand complexes and like charges destabilized them. A number of proteins - including lysozyme, superoxide dismutase, peroxidase, dextranase, carboxypeptidase A, ribonuclease A, and papain-produce useful charge ladders on acetylation; we are using these ladders to examine electrostatic influences on binding their substrates or ligands
21. This work was supported by NIH grant GM 51559. We thank J. Rao for helpful discussions and for the data involving vancomycin.