Similar Energetic Contributions of Packing in the Core of Membrane and Water-Soluble Proteins

Abstract

A major driving force for water soluble protein folding is the hydrophobic effect, but membrane proteins can not make use of this stabilizing contribution in the apolar core of the bilayer. It has been proposed that membrane proteins compensate by packing more efficiently. We therefore investigated packing contributions experimentally by observing the energetic and structural consequences of cavity creating mutations in the core of a membrane protein. We observed little difference in the packing energetics of water and membrane soluble proteins. Our results imply that other mechanisms are employed to stabilize the structure of membrane proteins.The hydrophobic effect is a major contributor to the stability of water soluble proteins, 1 but is essentially absent in the hydrocarbon core of a membrane. 2 Thus, the relative importance of other factors such as hydrogen bonding and van der Waals packing interactions must increase. While some hydrogen bonds can be key drivers of helix association, 3-5 we have argued that most appear to be modest contributors to tertiary structure stabilization in large membrane proteins. 2,6 If so, it implies that van der Waals packing dominates. Indeed, transmembrane helix dimers can be formed without any polar residues in the interface. 7 E-mail: bowie@mbi.ucla.edu. Supporting Information Available: Materials and methods, thermodynamic parameters for protein unfolding, X-ray diffraction data statistics, PDB codes for the mutant structures, and cavity measurements. This material is available free of charge via the Internet at http://pubs.acs.org. The lack of a clear consensus on packing density differences indicates that if they exist, it remains a rather subtle effect. NIH Public AccessGeometric analysis of static crystal structures is not always directly translatable to energetics, so we decided to probe packing contributions experimentally. Our analysis follows the classic work of Matthews and co-workers on the soluble protein T4 lysozyme. 12 In their work, the structural and energetic consequences of Leu-to-Ala substitutions in the protein core were investigated. The results are recapitulated in Fig. 1. They found a remarkably linear correlation between the change in thermodynamic stability and increased cavity size, in terms of both volume and surface area, created by the core substitutions. The extrapolated free energy at zero change in cavity size (1.9 kcal mol −1 ) provides an estimate of the change in the desolvation contribution alone (i.e. due to the hydrophobic effect), without a contribution from decreased packing. The slope of the lines (24 ± 3 cal mol −1 Å −3 and 20 ± 5 cal mol −1 Å −3 ) reflects the energetic cost of lost packing in the core of T4 lysozyme.To assess packing contributions in the core of a membrane protein, we made a set of large to small substitutions at buried residues in bacteriorhopsin (V49A, L94A, L111A, I148A, I148V and L152A) and obtained crystal structures for each of these mutants (see Table S1 in the Supporting Information). The most rel...

Stable folding core in the folding transition state of an α-helical integral membrane protein

“…Changes in stability seem to be related to changes in intramolecular interactions, presumably through packing effects and/or disruption of specific weak interactions such as the hydrogen bond between E204A and S194. These observations are in keeping with recent suggestions that packing energetics (9,43) and side-chain burial (44) play a key role in membrane protein stability. The destabilizing mutations we identified in helix G were subject to a Φ-value analysis to characterize the folding transition state.…”

Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh

“…increase in Ca ++ -permeability might be facilitated by the formation of a more flexible structure and thus the formation of a cavity, as shown for the L94A mutation of the lightdriven proton pump bacteriorhodopsin (compare Fig. 1) 21 . This cavity would be located in a hydrophobic patch as part of the conserved transmembrane helix three (TM3), only a helical turn apart from C128.…”

Structural differences between thermophilic and mesophilic membrane proteins

“…17 We have found that membrane proteins increase van der Waals packing contributions relative to soluble proteins by increasing the overall level of side-chain burial, rather than by improved packing efficiency (volume occupied by atoms). 18,19 Nevertheless, either increased packing efficiency or increased burial could be an important factor in stabilizing membrane proteins in hot environments. 15 We therefore investigated whether packing density and burial are different in mesophiles and thermophiles.…”