Fluorescence studies of rat cellular retinol binding protein II produced in Escherichia coli: an analysis of four tryptophan substitution mutants.
Rat intestinal cellular retinol binding protein II (CRBP II) is an abundant 134-residue protein that binds all-trans-retinol which contains 4 tryptophans in positions 9, 89, 107, and 110. Our ability to express CRBP II in Escherichia coli and to construct individual tryptophan substitution mutants by site-directed mutagenesis has provided a useful model system for studying the fluorescence of a multi-tryptophan protein. Each of the four mutant proteins binds all-trans-retinol with high affinity, although their affinities are less than that of the wild-type protein. Steady-state and time-resolved fluorescence analyses of these proteins indicate that W107 is at the hydrophobic binding site, W110 is in a polar environment, and the remaining two tryptophans are in a hydrophobic environment. Time-resolved fluorescence study indicates that excited-state energy transfer occurs from the hydrophobic tryptophans to W110. The Stern-Volmer analysis with acrylamide of these proteins reveals that static quenching occurs in the W9F mutant protein while others do not. The fluorescence of rat intestinal fatty acid binding protein (I-FABP), a related protein of known X-ray structure, was also studied for comparison. The results of these findings, coupled with those derived from NMR studies and molecular graphics, suggest that CRBP II undergoes minor structural changes in all of the mutant proteins. Since these effects may be cumulative on the protein structure and function, any conclusions derived from higher mutants in this family of proteins must be treated with caution.
Locke
B C
BC
Department of Chemistry, University of Chicago, Illinois 60637.
MacInnis
J M
JM
Qian
S
S
Gordon
J I
JI
Li
E
E
Fleming
G R
GR
Yang
N C
NC
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005454:Fluorescence Polarization; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012177:Retinol-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D054840:Retinol-Binding Proteins, Cellular; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics*; D014801:Vitamin A / Q000737:chemistry
D001059:Apoproteins; C518461:Rbp2 protein, rat; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D014801:Vitamin A; D014364:Tryptophan