Since the pioneering work of Anfinsen (1973), protein folding has been an area of major interest in life sciences. Although most studies focused on small globular proteins, significant advances were also achieved on membrane proteins (Booth et al., 2001). However, these results were achieved on a few transmembrane proteins whereas most of the others have been largely excluded from folding studies because they are considerably more difficult to work with (Booth et al., 2001). This is mainly due to the difficulties in mimicking the native, anisotropic environment of the biological membrane (Haltia and Freire, 1995; White et al., 2001). Recently, refined single-molecule unfolding experiments have allowed us to describe the folding process of single transmembrane helices and to estimate their folding kinetics (Kedrov et al., 2006a, 2004). The Na+/H+ antiporter NhaA from E. coli was chosen for these studies as it belongs to a large family of ion transporters ubiquitous in many prokaryots and eukaryots (Padan et al., 2001).
After imaging of the reconstituted NhaA molecules, proteins were partly unfolded by the AFM stylus leaving only two helices anchored in the lipid membrane (Figure 11.11 A). The corresponding force spectrum exhibited a characteristic sequence of peaks indicating that a single NhaA molecule was pulled from its C-terminal end (Kedrov et al., 2006a; Kedrov et al., 2004). In a next step, the
AFM stylus was lowered to close proximity of the membrane thereby relaxing the unfolded polypeptide chain (Figure 11.11A). At this stage, the system was left to equilibrate for 0.02 to 15 seconds allowing the coiled polypeptide chain to assume its free energy minimum. The efficiency of the refolding process was estimated from repeated folding and unfolding cycles of the molecule. Usually, all major peaks observed during the initial unfolding were detected with intensities similar to original ones if the refolding time delay lasted for >15 s. This indicated that the secondary structures of NhaA refolded and supports the postulation that unfolding and folding of transmembrane helices may be fully reversible (Hunt et al., 1997; Popot et al., 1987).
Spontaneous folding and insertion of a-helices into the hydrophobic core of a lipid bilayer was shown to be an energetically favorable process as the contacts between nonpolar aa residues and forcedly oriented molecules of the solvent decrease yielding an energy benefit of 20-40kBT per helix (Engelman et al., 1986). Significantly reducing the refolding time allowed determining in which order structural segments of the protein folded. Most obviously it was found that certain structural segments folded before others. In particular the ligand (sodium ion) binding site of the protein was the fastest folding element. After this the helices embedding the ligand-binding site followed. Then the adjacent helices followed. Figure 11.11B gives the folding kinetics determined for the individual structural segments.
In the near future we will be able to quantify the folding kinetics of membrane proteins in their native environment: the lipid membrane. These experiments include the controlled refolding in a wide range of timescales followed by detailed analysis of the consecutive unfolding traces. Plotting the refolding probability as a function of time allows determining the folding rate of the corresponding structural element (Carrion-Vazquez et al., 1999). These methods allow us to detect refolding intermediates and thereby reconstruct the folding pathway of an unfolded polypeptide into the functional protein (Kedrov et al., 2006a, 2004; Kessler et al., 2006). An important application of this approach will be the study of misfolded membrane proteins, that is, those trapped in alternative kinetically stable conformations.
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