Test of the Contribution of an Amino-Aromatic Hydrogen Bond to Protein Function

Robert S. Jamison, Bharati Kakkad, Daniel H. Ebert, Marcia E. Newcomer, David E. Ong

Research output: Contribution to journalArticlepeer-review

Abstract

Hydrogen bonds which form between a hydrogen bond donor and an aromatic ring as acceptor are thought to contribute to the stability and function of proteins. We have tested the function of such an interaction in a highly homologous pair of proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type II [CRBP(II)]. Both proteins bind the ligand all-trans-retinal with comparable affinities, but CRBP has an approximately 100-fold higher affinity for all-trans-retinol. The greater affinity of CRBP for all-trans-retinol has been attributed to the presence of an amino-aromatic hydrogen bond, which is absent in CRBP(II). We have generated a pair of mutant proteins, in which the amino-aromatic interaction was removed from CRBP and introduced into CRBP(II). Spectral analyses of retinol when bound to the wild-type and mutant CRBP suggested that it adopted an identical conformation within both proteins, a conformation that was distinct from that of retinol bound to CRBP(II), both wild-type and mutant. Unexpectedly, the affinities of the mutant binding proteins for all-trans-retinol were indistinguishable from those of their corresponding wild-type proteins. Further, in ligand competition experiments, there were no observable differences between mutant and wild-type CRBP, or between mutant and wildtype CRBP(II), in their preferences for binding all-trans-retinol versus all-trans-retinal. The results of this direct test of the proposed function of an amino role for such bonds, at least in this system.

Original languageEnglish (US)
Pages (from-to)11128-11132
Number of pages5
JournalBiochemistry
Volume34
Issue number35
DOIs
StatePublished - Sep 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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