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Protein Science (2004), 13:1670-1676. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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FOR THE RECORD

Replacement of proline with valine does not remove an apparent proline isomerization-dependent folding event in CRABP I

Lora L. Burns-Hamuro1,2, Paula M. Dalessio1 and Ira J. Ropson1

1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033, USA

(RECEIVED August 15, 2003; FINAL REVISION September 30, 2003; ACCEPTED September 30, 2003)



Abstract

Site-directed mutagenesis has frequently been used to replace proline with other amino acids in order to determine if proline isomerization is responsible for a slow phase during refolding. Replacement of Pro 85 with alanine in cellular retinoic acid binding protein I (CRABP-I) abolished the slowest refolding phase, suggesting that this phase is due to proline isomerization in the unfolded state. To further test this assumption, we mutated Pro 85 to valine, which is the conservative replacement in the two most closely related proteins in the family (cellular retinoic acid binding protein II and cellular retinol binding protein I). The mutant protein was about 1 kcal/mole more stable than wild type. Retinoic acid bound equally well to wild type and P85V-CRABP I, confirming the functional integrity of this mutation. The refolding and unfolding kinetics of the wild-type and mutant proteins were characterized by stopped flow fluorescence and circular dichroism. The mutant P85V protein refolded with three kinetic transitions, the same number as wild-type protein. This result conflicts with the P85A mutant, which lost the slowest refolding rate. The P85V mutation also lacked a kinetic unfolding intermediate found for wild-type protein. These data suggest that proline isomerization may not be responsible for the slowest folding phase of CRABP I. As such, the loss of a slow refolding phase upon mutation of a proline residue may not be diagnostic for proline isomerization effects on protein folding.

Keywords: proline isomerization; protein folding; folding intermediates; {beta}-sheet proteins; structural homology; stopped-flow kinetics, iLBP

Reprint requests to: Ira J. Ropson, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA; e-mail: iropson{at}psu.edu; fax: (717) 531-7072.

2 Present address: Provid Pharmaceuticals, 10 Knightsbridge Road, Piscataway, NJ 08854, USA.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03317804.


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