Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. SMFS unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding and refolding pathways. In contrast to various water-soluble proteins whose unfolding and refolding patterns have been characterized, only α-helical membrane proteins have been probed by SMFS. It was shown that α-helical membrane proteins unfold via many intermediates; this differs from the two-state unfolding process usually observed in water-soluble proteins. In membrane proteins, upon mechanically pulling the peptide end of the protein, single and grouped α-helices and polypeptide loops unfold in steps until the entire protein is unfolded. Whether the α-helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and the membrane. Each unfolding event relates to an unfolding intermediate with the sequence of these intermediates defining the unfolding pathway of the protein. β-barrel-forming membrane proteins are the second major group of membrane proteins and have not yet been studied by SMFS. To fill this void this study was designed to characterize interactions, unfolding, and refolding of the β-barrel forming outermembrane protein G (OmpG).Folding of transmembrane proteins, despite the important part these proteins play in every biological process in a cell, is studied in only a few examples. Of those only a handful were β-stranded membrane proteins (Tamm et al., 2004; Kleinschmidt et al., 2006). Current models describe that transmembrane β-barrels fold into the lipid membrane via two major steps. First the unfolded polypeptide interacts with the lipid surface where it then folds and inserts into the membrane (Kleinschmidt et al., 2006; Huysmans et al., 2010).
Conventionally, thermal or chemical denaturation is used to study folding of membrane proteins. In most cases membrane proteins were solubilized in detergent or exposed to urea to be studied, conditions that are not compatible with In vivo conditions. This suggests that the folding pathways described so far may not be a realistic representation of such pathways in nature. SMFS represents a unique approach to study the unfolding and refolding of membrane proteins into the lipid membrane (Kedrov et al., 2006; Kessler et al., 2006). Using SMFS makes it possible to study unfolding and refolding of membrane proteins in their nativephysiological environment with controlled pH, electrolyte, temperature, and most importantly in the absence of any chemical denaturant or detergent.
In this thesis, SMFS was utilized to unfold and refold OmpG in E coli lipid extract. Bulk unfolding experiments suggested that OmpG unfolds and folds reversibly and much faster than α-helical proteins (Conlan et al., 2000). The folding process is thought to be a coupled two-state membrane partition-folding reaction. To the contrary, the mechanical unfolding of OmpG consisted of many sequential unfolding intermediates. Our SMFS refolding experiments showed that a partially unfolded OmpG molecule also refolds via several sequential steps. The predominant refolding steps are defined by individual β-hairpins that could later assemble the transmembrane β-barrel of OmpG. In conclusion, the most probable unfolding and refolding pathways of OmpG as a membrane β-barrel protein go through the β-hairpins as the structural segments or unfolding-refolding intermediates and the process is a multi step one rather than the simple two state process.
We also used SMFS to study the physical interactions that switch the functional state and gating of OmpG. The structural changes that gate OmpG have been previously described by X-ray crystallography (Yildiz et al., 2006). They showed when the pH changes from neutral to acidic the flexible extracellular loop L6 folds into the pore and closes the OmpG pore. Here, SMFS was used to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At an acidic pH, a pH-dependent interaction at loop L6 was detected. This interaction changed the unfolding of loop L6 and β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were found to be unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pHdependent interactions change the folding of a peptide loop to gate the transmembrane pore. It has also been shown how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore and there are no global changes in protein conformation or mechanical properties. In the next step of interactions study, dynamic SMFS (DFS) was applied to quantify the parameters characterizing the energy barriers in energy landscape for unfolding of the OmpG.
Some of these parameters are: free energy of activation and distance of the transition state from the folded state. The pH-dependent functional switching of OmpG directs the protein along different regions at the unfolding energy landscape. The two functional states of OmpG sequential folding take the same unfolding pathway as β-hairpins I–IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a difference in Xu and κ in the energy landscape that translates to rigidified extracellular loop L6 at the gating area. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:14-qucosa-114947 |
| Date | 18 June 2013 |
| Creators | Damaghi, Mehdi |
| Contributors | Technische Universität Dresden, Fakultät Mathematik und Naturwissenschaften, Prof. Daniel Mueller, Prof. Dr. Stefan Diez, Prof. Dr. Petra Schwille |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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