Stabilizing and Destabilizing Clusters in the Hydrophobic Core of Long Two-stranded α-Helical Coiled-coils

Stanley C Kwok,Robert S Hodges

doi:10.1074/jbc.m401074200

Abstract

Detailed sequence analyses of the hydrophobic core residues of two long two-stranded alpha-helical coiled-coils that differ dramatically in sequence, function, and length were performed (tropomyosin of 284 residues and the coiled-coil domain of the myosin rod of 1086 residues). Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters. Subsequently, we designed a series of two-stranded coiled-coils to determine what defines a destabilizing cluster and varied the length of the destabilizing cluster from 3 to 7 residues to determine the length effect of the destabilizing cluster on protein stability. The results showed a dramatic destabilization, caused by a single Leu to Ala substitution, on formation of a 3-residue destabilizing cluster (DeltaT(m) of 17-21 degrees C) regardless of the stability of the coiled-coil. Any further substitution of Leu to Ala that increased the size of the destabilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (DeltaT(m) of 1.4-2.8 degrees C). These results suggested that the contribution of Leu to protein stability is context-dependent on whether the hydrophobe is in a stabilizing cluster or its proximity to neighboring destabilizing and stabilizing clusters.

Highlights

Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters
The two-stranded coiled-coil is an ideal model for such studies because its rod-like nature makes the folding problem a one-dimensional problem, thereby removing much of the complexity found in globular proteins [8]. Because this protein motif is widespread in nature (it was estimated that ϳ3% of all helical sequences are coiled-coils [9]), the coiled-coil and the ␣-helix have served extensively as models for the study of interactions in proteins, for example, hydrophobic interactions (10 –15), ionic interactions (16 –20), chain orientation [21,22,23], chain length effects [24],1 secondary structure propensities [25,26,27,28,29], context dependent interactions such as helix capping motifs (30 –33), and clustering of residues bilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (⌬Tm of 1.4 –2.8 °C)
Stabilizing and Destabilizing Clusters in Coiled-coils—In human skeletal myosin, 310 residues are involved in the hydrophobic core, of which 130 are destabilizing residues and 180 are stabilizing residues, based on our classification of stabilizing residues as Leu, Ile, Val, Met, Phe, and Tyr and destabilizing residues as Gly, Ala, Ser, Thr, Asn, Gln, Asp, Glu, Cys, Trp, His, Arg, and Lys

Summary

Introduction

The two-stranded coiled-coil is an ideal model for such studies because its rod-like nature makes the folding problem a one-dimensional problem, thereby removing much of the complexity found in globular proteins [8] Because this protein motif is widespread in nature (it was estimated that ϳ3% of all helical sequences are coiled-coils [9]), the coiled-coil and the ␣-helix have served extensively as models for the study of interactions in proteins, for example, hydrophobic interactions (10 –15), ionic interactions (16 –20), chain orientation [21,22,23], chain length effects [24],1 secondary structure propensities [25,26,27,28,29], context dependent interactions such as helix capping motifs (30 –33), and clustering of residues bilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (⌬Tm of 1.4 –2.8 °C).

Objectives

Results

Conclusion