Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily.


Abstract

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures. Study holds ProTherm entries: 12068, 12069, 12070, 12071, 12072, 12073, 12074, 12075, 12076, 12077, 12078 Extra Details: protein stability; mutagenesis; evolution; conserved residues; lipocalins;,protein folding; serum retinol-binding protein; crystal structures

Submission Details

ID: ikoDyUuB4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:43 p.m.

Version: 1

Publication Details
Greene LH;Chrysina ED;Irons LI;Papageorgiou AC;Acharya KR;Brew K,Protein Sci. (2001) Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily. PMID:11604536
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AQB 1997-07-28T00:00:00+0000 1.65 RETINOL-BINDING PROTEIN (RBP) FROM PIG PLASMA
1ERB 1993-10-01T00:00:00+0000 1.9 THE INTERACTION OF N-ETHYL RETINAMIDE WITH PLASMA RETINOL-BINDING PROTEIN (RBP) AND THE CRYSTAL STRUCTURE OF THE RETINOID-RBP COMPLEX AT 1.9 ANGSTROMS RESOLUTION
1FEL 1994-08-29T00:00:00+0000 1.8 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1FEM 1994-08-29T00:00:00+0000 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1FEN 1994-08-29T00:00:00+0000 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1HBP 1993-02-05T00:00:00+0000 1.9 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1HBQ 1993-02-05T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1KT3 2002-01-15T00:00:00+0000 1.4 Crystal structure of bovine holo-RBP at pH 2.0
1KT4 2002-01-15T00:00:00+0000 1.46 Crystal structure of bovine holo-RBP at pH 3.0
1KT5 2002-01-15T00:00:00+0000 1.46 Crystal structure of bovine holo-RBP at pH 4.0

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Retinol-binding protein 4 P02753 RET4_HUMAN
100.0 Retinol-binding protein 4 P61641 RET4_PANTR
94.5 Retinol-binding protein 4 M5AXY1 RET4_FELCA
94.5 Retinol-binding protein 4 Q28369 RET4_HORSE
92.9 Retinol-binding protein 4 P27485 RET4_PIG
92.3 Retinol-binding protein 4 P18902 RET4_BOVIN
91.8 Retinol-binding protein 4 P06912 RET4_RABIT