MEDIOS AUXILIARES
B) Pozos y saneamientos
7.1.29. Instalación de tuberías en el interior de zanjas
Most approaches to study insertion and folding mechanism of membrane proteins have been carried on α-helical proteins. Wimley and White established a scale based on amino acid hydrophobicity by measuring the partitioning of small peptides into the membrane inter-phase (Wimley and White, 1996). Later on, von Heijne and co- workers tested the capability of different peptide segments to adopt a transmembrane topology using an in vitro translation system. Based on these findings, they proposed a model for insertion and folding of α-helical membrane proteins (chapter 1). Meanwhile, investigations carried on β-barrel proteins led to proposing the coupled insertion and folding model (Tamm et al., 2004; Huysmans et al, 2010).
SMFS studies of folding mechanism of membrane proteins are limited to BR and NhaA (Kedrov et al., 2004; Kedrov et al., 2006; Kessler et al, 2006). The refolding of these proteins initiated from partially unfolded proteins, i.e. the last structural segment kept into the membrane, which makes it proper to study the refolding of the unfolded peptide refolding inside the membrane. However, the weak point would be missing the insertion part and also interference of surrounded proteins in the folding procedure.
The mechanisms that guide the folding and unfolding of Omps are of pertinent interest (White and Wimmely, 1999; Tomm et al., 2004; Kleinschmidt et al., 2002). So far, most experiments investigating the folding of Omps have first denatured Omps in detergent or urea and then characterized the refolding process into a lipid bilayer or detergent (Conlan et al., 2003; Hong et al., 2007). Such bulk unfolding experiments suggest that OmpG unfolds and refolds reversibly. The folding process of Omps is described as being coupled with membrane insertion (Tamm et al., 2004). Folding and the insertion process of β-barrel transmembrane protein PagP from E. coli has been recently described (Huysmans et al., 2010). PagP, solubilized and denatured in 10 M urea, was found to adsorb to the lipid head groups of the bilayer, where it forms a transition state that tilts and inserts into the lipid membrane to complete the folding process. However, these models contrast with the SMFS results, in which single OmpG molecules in a buffer solution were mechanically stressed to induce unfolding from the native lipid membrane (Sapra et al., 2009). These single-molecule
experiments clearly show that OmpG molecules unfold via many sequential unfolding intermediates, and detailed unfolding pathways were described. The unfolding step of a single β-hairpin characterizes the transition from one unfolding intermediate to the next. However, the difference between chemical denaturation and refolding and mechanical unfolding experiments may be due to a different reason. First, it may be assumed that the refolding mechanism in the absence of external force does not reflect unfolding under an applied force. Second, the experimental conditions may alter the unfolding and folding pathways taken by β-barrel membrane proteins. In the case of α-helical transmembrane proteins, it has been shown that alterations in the temperature and buffer solution, within the physiologically relevant range, can considerably modify unfolding pathways (Kedrov et al., 2007). Therefore, it is not surprising that exposure of membrane proteins to urea, detergents and mechanical stress may force them along very different unfolding and folding pathways.
SMFS experiments showed that a partially unfolded OmpG molecule refolds via many sequential steps. The predominant refolding steps are defined by individual β- hairpins that later assemble to the transmembrane β-barrel of OmpG. Each β-hairpin shows intrinsic folding kinetics, and these kinetic parameters determine the refolding pathway of that the OmpG polypeptide will take. It is interesting to investigate which factors (such as different environment or biological chaperones) influence the folding kinetics and, thus, the folding hierarchy of a transmembrane β-barrel. It would also be interesting to study the effects of various lipid compositions on refolding kinetics. Another approach would be to study the role of adjacent proteins on the refolding process and kinetics of target protein. This can open an era in single protein-protein interaction studies.
Nevertheless, the main goal would be to understand the folding mechanism of β- barrel proteins inside bacteria or mitochondria. To mimic in vivo conditions, a new SMFS experiment was designed in order to study the refolding of single β-barrel protein in the presence of β-barrel assembly machinery. To do so, β-barrel assembly machinery (BAM) complex reconstituted in a membrane would be adsorbed on the Mica and the β-barrel protein will be picked from different patches on the same support. This required a complicated movement of AFM piezo, which is now possible by new software that was designed specifically for this task by JPK.
This software adds an extra layer of control on the piezo movement in the microscope set up that enables us to pick a protein from a spot on the mica and move it to a different position10, i.e. in this case, where the BAM complex is located. Using this
new SMFS design makes it also possible to study the mechanism of membrane protein insertion and folding into the membrane bilayer much more representative to in vivo conditions (Figure 5.1).
Figure 5.1Schematicdemonstration of the new SMFS experiment on membrane protein folding design: This new technique enables us to study the folding mechanism of a single membrane protein in
different membrane compositions, with the presence of chaperons, and with the presence of other membrane proteins or large complexes.
Another option is to modify the AFM tip and covalently attach the protein onto the tip via a linker such as polyethylene glycol (PEG) molecule. The linker will reduce the interference of the AFM tip and the protein while inserting or folding inside the membrane. One of the difficulties of this experiment is the aggregation of membrane protein in aqueous solutions. Furthermore, it should be ensured that only interactions of single proteins are measured. The experimental procedure would be exactly the same as what we did in normal refolding experiments (described in chapter 3). In
10 It used to be impossible to pull from a position on the sample and move it
brief, we would approach the modified tip to the membrane and held it for a certain amount of time close to the membrane surface to allow the unfolded polypeptide to bind, insert, and fold into the bilayer. By withdrawing the cantilever in different time scales, we can watch any of the three mentioned steps.
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