About This Project
We will use a facile protein engineering approach to a) convert membrane-bound protein methane monooxygenase particulate (pMMO) into the water-soluble QTY analog (pMMO-QTY), and b) produce water-soluble pMMO-QTY at kilogram scale in filamentous fungus Trichoderma reesei and Myceliophthora thermophile systems (not in E.coli). QTY (Glutamine-Threonine-Tyrosine) replaces Leucine, Isoleucine/Valine and Phenylalanine, respectively, in membrane-spanning domains of pMMO to achieve water-solubility.
Ask the Scientists
Join The DiscussionMotivating Factor
Methane (CH4) emissions account for ca. 30% of global warming (IEA 2022 Global Methane Tracker, IEA, Paris: https://www.iea.org/reports/gl...). Natural CH4 sources may increase via a feedback loop to warming. Climate change could be mitigated by new technologies for oxidizing atmospheric CH4, area emissions, and various point sources. Higher concentrations (>44,000 ppm) can be flared but some 75% of the pollution is composed of smaller contributions and cannot be oxidized to scale with existing technologies.
Methane monooxygenase (MMO) enzymes found in methanotrophic bacteria naturally catalyze CH4 to methanol conversion in a one-step reaction under ambient conditions. Particulate MMO (pMMO) is a membrane-bound enzyme that catalyzes the same conversion. Oxidation of dilute CH4 may be possible using methanotrophs or cell-free MMO in flow-through reactors or via plant expression. However, manipulation of pMMO that has the highest affinity for CH4 is considered very difficult.
Specific Bottleneck
It is notoriously difficult to express, purify and study membrane proteins because they require detergent to stabilize them after removing the proteins from the cell membrane. Therefore, it is impossible to produce membrane proteins in industrial scale in kilograms and tons. Thus, innovative methods must be found to express and purify membrane proteins in large scale.
1) Using a QTY code (amino acid L->Q, I/V->T, F->Y conversion) we will engineer the membrane protein enzymes particulate methane monooxygenases pMMO (Bath) into water-soluble analogs pMMO-QTY such that the enzyme retains catalytic function to convert methane to methanol. Availability of a water-soluble form overcomes current bottlenecks associated with non-functional or low-activity pMMO assembled outside the native host or pMMO of near-full function expressed outside its native host.
2) We will produce water-soluble pMMO-QTY variant at industrial scale (kg/tons) at affordable cost.
Actionable Goals
We invented a simple QTY code to convert a hydrophobic alpha-helix to a hydrophilic one. We demonstrated that this code can convert membrane proteins such as G protein-coupled receptors (GPCRs) and cytokine receptors into their water-soluble analogs that retain their biological function, i.e. ligand binding affinities similar to native receptors (Cover Image).
Water-soluble QTY analog receptor CXCR4-QTY was used to design a highly sensitive detection device. Structural bioinformatic studies using AlphaFold2 of integral membrane proteins were carried out. Thus, water-soluble QTY variant superposed very well with the structure corresponding to native membrane proteins and RMSDs of often less than 2Å including GPCRs, glucose transporters, SLC solute carrier transporters, ABC transporters, glutamate transporters and monoamine transporters.
We will now apply this approach to express water-soluble pMMO trimers and achieve high density arrays using immobilization on S-layer surfaces.
Budget
BUDGET (12-MONTH PERIOD)
PERSONNEL: Salaries $88,832, Benefits $24,162, Total $112,994
GENERAL: CORES Services $12,000, Materials and Supplies $11,370, Total $23,370
TOTAL DIRECT: $136,364
TOTAL F&A (10%): $13,636
PROJECT TOTAL: $150,000
JUSTIFICATION
Martin Egli, PhD, Professor/PI. 1.5 calendar month effort. He will head the efforts to convert membrane-bound pMMO into water-soluble pMMO-QTY and will liaise with Dr. Shuguang Zhang at MIT.
Li Lei, MD, Assistant in Biochemistry. 4 calendar month effort. She will express and purify pMMO-QTY-variant in baculovirus-Sf9 insect cell, fungal and thermophilic systems and conduct activity assays.
Joel Harp, PhD, Research Assistant Professor. 4 calendar month effort. He is an experienced structural biologist and biochemist. He will ascertain by cryo-EM that parent pMMO and pMMO-QTY variant adopt similar folds.
Materials: Chemicals, cell culture, columns, solvents, glass- and plasticware, EM grids, core and fee for service charges.
Meet the Team
Team Bio
Joel M. Harp PhD, Biomed. Sci., Univ. of TN, Knoxville, 2000. Currently employed as Res. Asst. Prof., Egli lab, VU. Expertise in macromolecular X-ray and neutron crystallography, and electron microscopy.
Li Lei MD, Luzhou Med. College, Luzhou, China, 1981. Currently employed as Asst. in Biochemistry, Egli lab, VU. Expertise in protein expression in bacterial and insect cells as well as mammalian systems, protein engineering, purification, activity assays, and all crystallization techniques.
Martin Egli
A. Education and Training
ETH-Zürich, Switzerland | Diploma | 09/1984 | Chemistry |
ETH-Zürich, Switzerland | Ph.D. | 10/1988 | Chem. Crystallography |
MIT, Cambridge MA | Postdoc | 03/1992 | Structural Biology |
B. Employment and Positions
2005 - Professor, Dept. of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN
2003-2004 Assoc. Prof., Dept. of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN
2000-2003 Assoc. Professor, Dept. of Biological Sciences, Vanderbilt University, Nashville, TN
1995-1999 Asst. Professor, Dept. of Molec. Pharm. & Biol. Chem., Northwestern University, Chicago, IL
1992-1995 Lecturer and Habilitand, Organic Chemistry Laboratory, ETH-Zürich, Zürich, Switzerland
1989-1992 Postdoctoral Fellow, Prof. Alexander Rich, Dept. of Biology, M.I.T., Cambridge, MA
1985-1988 Ph.D. student, advisors Profs. Jack D. Dunitz and Vladimir Prelog – Nobelist Chemistry 1975, Organic Chemistry Laboratory, ETH-Zürich, Zürich, Switzerland
C. Selected Honors
2023 Elected as member of the European Academy of Sciences and Arts
2013 Alexander Rich Award, Dept. of Biology, MIT
2011-2013 WCU Professor, Biophysics & Chem. Biol. Department, Seoul National University, Seoul, Korea
2009 Elected AAAS Fellow (Chemistry section)
2006 Visiting Professor, Dept. of Chemistry, Oxford University, Oxford, UK
2005 Visiting Professor, Dept. of Chemistry, Seoul National University, Seoul, Korea
2005 Vanderbilt Chancellor Award for Research (KaiABC circadian clock)
D. Contributions to Science
Published work to date includes some 300 refereed papers and reviews and two books: Egli, M., Herdewijn, P., Eds., Chemistry and Biology of Artificial Nucleic Acids, Wiley-VCH Publishers, Weinheim, Germany, 2012; and Blackburn, G.M., Egli, M., Gait, M.J., Watts, J.K., Eds., Nucleic Acids in Chemistry and Biology, 4. ed., R. Soc. Chem., Cambridge, UK, 2022.
Additional Information
FIGURE 1. Cryo-ET STA of native pMMO trimer from isolated ICMs and S-layer 2D lattice surface with specific linker Fc domain of IgG fused to pmoC of pMMO with extremely high-density array. a) A tomographic slice of isolated ICM. A yellow dashed circle encloses a unit of 7 particles. b) Sub-tomogram average of the 7-particle unit. c) An enlarged view of the central particle, 3 trimer pMMO in (b), superimposed with the docked crystal structure of the pMMO trimer (PDB 3RGB). d) The pMMO trimer STA map at 4.8 Å resolution, with each pMMO monomer consisting of PmoA (pink), PmoB (blue), and PmoC (cyan). e) A central slice view of pMMO map superimposed with pMMO crystal structure. f) Overall structure of active pMMO trimer, with PmoA, PmoB, and PmoC colored as in panel d. g, h) Side and cross-sectional views of the pMMO map, superimposed with the cryo-EM pMMO (PDB 7S4H). i) Three invariant residues, N227, H231, and H245 from PmoC helices γ5 and γ6, bind copper in the cryo-EM structure of pMMO (PDB 7S4H). j) Illustration of knights without S-layer surface, k) S-layer 2D lattice proteins organize knights (receptors, etc.) with 100% upward orientation. l) SEM image of S-layer surface on bacterial membrane. m) high-resolution image of S-layer with 13nm square lattice. n) a receptor CXCR4QTY fused to Fc of IgG that bind to protein A on designed S-layer surface. o) The S-layer has an extremely high surface density, 1 trillion/cm2 (1012/cm2). (Fig. 1a-i is adopted from ref. [3]).
FIGURE 2. Conversion of PMOA to its water soluble PMOA-QTY analog (similar for PMOB and PMOC).
Selected citations from Solution Statement with links to journals:
1. Lieberman RL, Rosenzweig AC. Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature 2005; 434(7030):177-182. This paper reports the crystal structure of pMMO.
2. Chang WH, et al. Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation. J Am Chem Soc. 2021;143(26):9922-9932. This paper reports the cryo-EM structure of pMMO.
3. Zhu Y, et al. Structure and activity of particulate methane monooxygenase arrays in methanotrophs. Nat Commun. 2022;13(1):5221. This paper investigates pMMO high-density arrays and their higher enzymatic catalytic activities.
4. Zhang S, Egli M (2022) Hiding in plain sight: three chemically distinct α-helix types. Quarterly Review of Biophysics (QRB) 55, e7. This paper explains the molecular basis of how and why the QTY code works.
5. Zhang S, et al. (2018) QTY code enables design of detergent-free chemokine receptors that retain ligand-binding activities. Proc. Natl. Acad. Sci. USA 115 (37) E8652-E8659. PMID: 30154163. This paper marks the first report of the QTY code.
6. Li M, et al. (2024) Design of a water-soluble transmembrane receptor kinase with intact molecular function by QTY code. Nature Communications 15(1), 4293. PMID: 38858360. This paper inspired us to engineer water-soluble pMMO-QTY variant.
7. Sleytr UB, Schuster B, Egelseer EM, Pum D. S-layers: principles and applications. FEMS Microbiol Rev. 38(5):823-864 (2014). PMID: 24483139. This paper summarizes the S-layer protein 2D lattice for various applications.
8. Ferner-Ortner-Bleckmann J, et al. Surface-layer lattices as patterning element for multimeric extremozymes. Small. 2013; 9(22):3887-3894. PMID: 23757161. This paper describes how to make 2D enzyme arrays on S-layer proteins.
Project Backers
- 0Backers
- 0%Funded
- $0Total Donations
- $0Average Donation