Role of Local Sequence in the Folding of Cellular Retinoic Acid Binding Protein I:  Structural Propensities of Reverse Turns

Abstract

The experiments described here explore the role of local sequence in the folding of cellular retinoic acid binding protein I (CRABP I). This is a 136-residue, 10-stranded, antiparallel beta-barrel protein with seven beta-hairpins and is a member of the intracellular lipid binding protein (iLBP) family. The relative roles of local and global sequence information in governing the folding of this class of proteins are not well-understood. In question is whether the beta-turns are locally defined by short-range interactions within their sequences, and are thus able to play an active role in reducing the conformational space available to the folding chain, or whether the turns are passive, relying upon global forces to form. Short (six- and seven-residue) peptides corresponding to the seven CRABP I turns were analyzed by circular dichroism and NMR for their tendencies to take up the conformations they adopt in the context of the native protein. The results indicate that two of the peptides, encompassing turns III and IV in CRABP I, have a strong intrinsic bias to form native turns. Intriguingly, these turns are on linked hairpins in CRABP I and represent the best-conserved turns in the iLBP family. These results suggest that local sequence may play an important role in narrowing the conformational ensemble of CRABP I during folding.

Natural polypeptide scaffolds: β‐sheets, β‐turns, and β‐hairpins

“…21,31 Also critical are interactions within the turn residues: for example, the type I -turn with a G1 bulge is emerging as a common feature in many stable peptide hairpins. [32][33][34][35][36] Based on examples studied to date, stability requires strand lengths of five or more residues. 37 …”

Local sequence information in cellular retinoic acid‐binding protein I: Specific residue roles in β‐turns*

“…24 CD spectra of the turn fragments were examined at concentrations from 10 M to 1 mM, and with one exception (the Gly78Ala variant of turn IV, see below), there was no indication of concentration dependence in the observed conformational ensemble of the fragments.…”

“…24 The pH was adjusted with 6M HCl, and reported pH is uncorrected for the deuterium isotope effect. In samples containing urea, solid ultrapure urea d 4 (CIL, Andover, MA) was added to a concentration of 7M prior to final pH adjustment.…”

“…24 CRABP I is 136 amino acids in length and is made up of a 10-stranded, up-down, ␤-barrel with a short helix-turn-helix between strands 1 and 2 of the barrel (Figure 1). 25 CRABP I samples several kinetic intermediates in its folding, 26 and thus represents a different and less extensively studied class of proteins than the small two-state folding proteins (Ͻ100 amino acids) previously shown to have locally defined turns.…”

A career pathway in protein folding: From model peptides to postreductionist protein science

“…Our early studies have focused on a heterologous protein expressed in E. coli, and we chose to use cellular retinoic acid-binding protein I (CRABP I) initially because of our extensive experience with the folding of this b-rich protein in vitro. [51][52][53][54][55][56] A clear advantage of using a non-E. coli protein is that folding can be studied without altering directly the cellular physiology by virtue of the function of the protein under study. CRABP I is also delicately balanced between successful folding and formation of aggregates, both in vitro and in vivo.…”