An Unfolding Story of Helical Transmembrane Proteins

Abstract

Reversible unfolding of helical transmembrane proteins could provide valuable information about the free energy of interaction between transmembrane helices. Thermal unfolding experiments suggest that this process for integral membrane proteins is irreversible. Chemical unfolding has been accomplished with organic acids, but the unfolding or refolding pathways involve irreversible steps. Sodium dodecyl sulfate (SDS) has been used as a perturbant to study reversible unfolding and refolding kinetics. However, the interpretation of these experiments is not straightforward. It is shown that the results could be explained by SDS binding without substantial unfolding. Furthermore, the SDS perturbed state is unlikely to include all of the entropy terms involved in an unfolding process. Alternative directions for future research are suggested: fluorinated alcohols in homogeneous solvent systems; inverse micelles; and fragment association studies.Genomic sequencing indicates that integral membrane proteins are abundant, and there are numerous examples of disease states which result from membrane protein misfolding (1). These ought to be sufficient incentives to study the folding of integral membrane proteins, but some biochemists have been discouraged by the notorious difficulty of determining membrane protein 3D structures and the restrictions of working with detergent or vesicle systems. However, the outlook in this field has brightened, as demonstrated in several recent reviews. Bowie (2) has provided an excellent summary of the main issues in current membrane protein folding research, and MacKenzie (3) recently comprehensively surveyed the folding and stability of helical membrane proteins. I will restrict my comments here to a critical evaluation of some past and recent attempts to reversibly unfold helical integral membrane proteins, followed by some suggestions for promising future research directions.

Buried water molecules in helical transmembrane proteins

“…The resulting mutants should be less stable to unfolding conditions, as has been shown for water-soluble proteins (Griffin et al 2002;Takano et al 2003). Unfortunately, a methodology for unequivocal reversible unfolding of helical TM proteins is not available (Renthal 2006). Nevertheless, mutations which cause misfolding are known for some membrane proteins.…”

From Valleys to Ridges: Exploring the Dynamic Energy Landscape of Single Membrane Proteins

“…The folding of transmembrane proteins is a particularly long-standing problem, and relatively little biophysical information on their energy landscapes is available. [116][117][118] In the past years, different SMFS techniques were combined with kinetic models [61] and fluctuation theorems [119] to reveal insights into the energy landscapes of proteins. Due to the possibility of detecting single-molecule events, force spectroscopy can detect co-existing folding and unfolding pathways and non-accumulative intermediates populating multi-dimensional energy landscapes of biological molecules.…”

The transition state for integral membrane protein folding

“…A small chaotrope such as urea gives relatively unstructured states for water-soluble proteins. In contrast, the detergent SDS is used here, resulting in only partial denaturation and the interactions between SDS and bR during unfolding are poorly understood (17). SDS is an anionic surfactant with a long hydrocarbon tail and is a bulky denaturant with physical properties that are quite different from those of urea.…”