Thermodynamics of Mistic protein folding
Final Report Abstract
This project was aimed at elucidating the folding thermodynamics of the prokaryotic membrane protein Mistic. We reasoned that this four-span, 110-residue membrane protein from Bacillus subtilis might be an ideal candidate for studying membrane-protein folding in vitro because it is only marginally hydrophobic, self-inserts into lipid bilayers independently of the translocon, and assists in the recombinant overexpression and bilayer insertion of other membrane proteins. The principal goals at the onset of this report period were to (i) provide a quantitative comparison of the conformational stability of Mistic among different membrane-mimetic systems to enable a systematic dissection of factors such as hydrophobic match/mismatch, importance of polar headgroup vs. hydrophobic tail interactions, effects of “harsh” vs. “mild” detergents, and reconstructive denaturation without the ambiguity inherent in other experimental assays of membrane-protein stability; (ii) obtain insights at the amino acid level into the ability of Mistic to self-insert spontaneously and in a translocon-independent way into artificial lipid bilayers and biological membranes; and (iii) design and produce a monomeric water-soluble variant of Mistic for a direct comparison of the thermodynamics and kinetics of protein folding between membrane-mimetic and aqueous environments. As anticipated, the experimental approaches devised for addressing these goals required extensive methodological development and optimisation. In particular, we first quantified the effects of urea on micellar properties to account for the decrease in both the number and the size of micelles upon addition of urea to a protein suspended in detergent solution. Next, we introduced and validated automated circular dichroism spectroscopy as a powerful method for performing quantitative protein-unfolding titrations with enhanced throughput and unparalleled precision. This was essential because we later aimed at dissecting incremental changes in protein stability across a wide range of membrane-mimetic systems, which oftentimes differed from one another by as little as a single methylene group in the alkyl chain making up the hydrophobic phase embedding the protein. Finally, we established size-exclusion chromatography coupled to triple detection by absorbance spectroscopy, static light scattering, and refractive index detection to demonstrate that urea-unfolded Mistic and detergent micelles are not physically associated with each other under denaturing conditions. Here, a new protocol for data analysis became necessary because Mistic happened to co-elute with an important standard detergent in spite of being detached from detergent micelles at high urea concentrations. Close investigation of this issue was mandatory because detergent interactions of the unfolded protein would have invalidated our approach relying on the availability of a micelle-detached unfolded state serving as a reference common to all unfolding/refolding equilibria. Once all of the above prerequisites were met, we tackled the first goal by determining the structure and dynamics of the detergent-free urea-unfolded state of Mistic and, additionally, elucidating the “downhill” unfolding process in the presence of the only detergent that did not support two-state folding. For all other membrane-mimetic systems studied, a thermodynamic approach based on a classical two-state protein-folding scenario was very fruitful and yielded surprising insights into the forces that stabilise Mistic at the hydrophobic/hydrophilic interface. In particular, we found that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilise Mistic much more efficiently than nonpolar interactions with the micelle core. These experiments unveiled the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalised the differential suitability of detergents for the extraction and solubilisation of Mistic-tagged membrane proteins, thus having implications for the recombinant production of integral membrane proteins. Although Mistic is a two-state folder under equilibrium conditions, the kinetics of insertion and folding went far beyond two-state behaviour and turned out to be so complex as to invalidate traditional approaches based on Φ-value analysis, as envisaged in the second of the above goals. By contrast, the third goal was more straightforward to achieve in that we succeeded in transferring our results on individual helical peptide fragments obtained during the first funding period to the protein level. Specifically, we engineered a single-point mutant dubbed Mistic(I41N) that is soluble in aqueous solution in the absence of a membrane-mimetic environment. The finding that this protein variant is unstructured and becomes insoluble upon structure induction by protecting osmolytes supports the conclusion that membrane properties such as the interfacial dipole potential play an essential role in stabilising the native state of the unconventional membrane protein Mistic.
Publications
- Monitoring detergent-mediated solubilization and reconstitution of lipid membranes by isothermal titration calorimetry. Nat. Protoc. 2009, 4, 686–697
Heerklotz, H.; Tsamaloukas, A. D.; Keller, S.
- Membrane solubilisation and reconstitution by octylglucoside: comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry. Biophys. Chem. 2010, 150, 105–111
Krylova, O. O.; Jahnke, N.; Keller, S.
- Membrane translocation assayed by fluorescence spectroscopy. Methods Mol. Biol. 2010, 606, 271–289
Broecker, J.; Keller, S.
- Nonlinear least-squares data fitting in Excel spreadsheets. Nat. Protoc. 2010, 5, 267–281
Kemmer, G.; Keller, S.
- Protein folding in membranes. Cell. Mol. Life Sci. 2010, 67, 1779–1798
Fiedler, S.; Broecker, J.; Keller, S.
- α-Helical transmembrane peptides: a “divide and conquer” approach to membrane proteins. Chem. Phys. Lipids 2010, 163, 1–26
Bordag, N.; Keller, S.
- Characterization of membrane proteins in isolated native cellular membranes by dynamic nuclear polarization solid-state NMR spectroscopy without purification and reconstitution. Angew. Chem. Int. Ed. 2012, 51, 432–435
Jacso, T.; Franks, W. T.; Rose, H.; Fink, U.; Broecker, J.; Keller, S.; Oschkinat, H.; Reif, B.
(See online at https://doi.org/10.1002/anie.201104987) - New peptide characterized in reversible oligomerization of the peptide, which is controlled by a pH switch, useful as protein affinity tag for the purification of protein. EP 2423217-A1; published 29th February 2012
Bordag, N.; Keller, S.
- Automated circular dichroism spectroscopy for mediumthroughput analysis of protein conformation. Anal. Chem. 2013, 85, 1868–1872
Fiedler, S.; Cole, L.; Keller, S.
(See online at https://doi.org/10.1021/ac303244g) - Impact of urea on detergent micelle properties. Langmuir 2013, 29, 8502–8510
Broecker, J.; Keller, S.
(See online at https://doi.org/10.1021/la4013747) - The mechanism of denaturation and the unfolded state of the α-helical membrane-associated protein Mistic. J. Am. Chem. Soc. 2013, 135, 18884–18891
Jacso, T.; Bardiaux, B.; Broecker, J.; Fiedler, S.; Baerwinkel, T.; Mainz, A.; Fink, U.; Vargas, C.; Oschkinat, H.; Keller, S.; Reif, B.
(See online at https://doi.org/10.1021/ja408644f) - Mistic’s membrane association and its assistance in overexpression of a human GPCR are independent processes. Protein Sci. 2014
Marino, J.; Bordag, N.; Keller, S.; Zerbe, O.
(See online at https://doi.org/10.1002/pro.2582) - Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic. J. Am. Chem. Soc. 2014, 136, 13761–13768
Broecker, J.; Fiedler, S.; Gimpl, K.; Keller, S.
(See online at https://doi.org/10.1021/ja5064795) - Real-time monitoring of membrane-protein reconstitution by isothermal titration calorimetry. Anal. Chem. 2014, 86, 920–927
Jahnke, N.; Krylova, O. O.; Hoomann, T.; Vargas, C.; Fiedler, S.; Pohl, P.; Keller, S.
(See online at https://doi.org/10.1021/ac403723t)
