Test of the contribution of an amino-aromatic hydrogen bond to protein function.
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 retinal. 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 wild-type 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-aromatic hydrogen bond did not support a functional role for such bonds, at least in this system.
Jamison
R S
RS
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA.
Kakkad
B
B
Ebert
D H
DH
Newcomer
M E
ME
Ong
D E
DE
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers / Q000235:genetics; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051381:Rats; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054840:Retinol-Binding Proteins, Cellular; D013053:Spectrophotometry; D013237:Stereoisomerism; D014801:Vitamin A / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D008024:Ligands; C518461:Rbp2 protein, rat; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D014801:Vitamin A; D012172:Retinaldehyde