Bacteria that oxidize methane to methanol are central to mitigating emissions of methane, a potent greenhouse gas. The nature of the copper active site in the primary metabolic enzyme of these bacteria, particulate methane monooxygenase (pMMO), has been controversial owing to seemingly contradictory biochemical, spectroscopic, and crystallographic results. We present biochemical and electron paramagnetic resonance spectroscopic characterization most consistent with two monocopper sites within pMMO: one in the soluble PmoB subunit at the previously assigned active site (CuB) and one ~2 nanometers away in the membrane-bound PmoC subunit (CuC). On the basis of these results, we propose that a monocopper site is able to catalyze methane oxidation in pMMO.
Supplementary Information includes Supplementary methods, 5 supplementary figures, 3 supplementary tables, and supplementary references. Supplementary MethodsΔΔG calculation. The ΔΔG of the 830 mutants in the quantitative dataset can be calculated in two ways: (1) by taking the difference between WT and mutant fitted ΔG(H 2 O) values given by the linear extrapolation method 1 (ΔΔG(H 2 O)), or (2) by taking the difference between WT and mutant C m values and multiplying by their mean m-value 2 (ΔΔG(m-avg)). The ΔΔG(m-avg) value avoids fitting issues near low denaturant values when estimating ΔG(H 2 O), but loses validity when variants greatly affect the m-value or the stability of the mutant protein 2,3 . As roughly 80% of the dataset has values ±1 kcal/mol away from WT, and the WT m-value is within 1 standard deviation of the mean m-value for the dataset, the m-avg method appears valid. After averaging repeat measurements, the two methods correlate extremely well (r = 0.99) with over 90% of the dataset differing by no more than 0.2 kcal/mol and none more than 1 kcal/mol ( Supplementary Fig. 2a). The only major difference between the methods is that the ΔΔG(H 2 O) method is less precise than the ΔΔG(m-avg) method ( Supplementary Fig. 2b), likely due to the uncertainties inherent in estimating ΔG(H 2 O) values. Thus, the mean of the ΔΔG(m-avg) values recorded for each variant (referred to as ΔΔG) were used for all further analysis.
The stability, activity, and solubility of a protein sequence are determined by a delicate balance of molecular interactions in a variety of conformational states. Even so, most computational protein design methods model sequences in the context of a single native conformation. Simulations that model the native state as an ensemble have been mostly neglected due to the lack of sufficiently powerful optimization algorithms for multistate design. Here, we have applied our multistate design algorithm to study the potential utility of various forms of input structural data for design. To facilitate a more thorough analysis, we developed new methods for the design and high-throughput stability determination of combinatorial mutation libraries based on protein design calculations. The application of these methods to the core design of a small model system produced many variants with improved thermodynamic stability and showed that multistate design methods can be readily applied to large structural ensembles. We found that exhaustive screening of our designed libraries helped to clarify several sources of simulation error that would have otherwise been difficult to ascertain. Interestingly, the lack of correlation between our simulated and experimentally measured stability values shows clearly that a design procedure need not reproduce experimental data exactly to achieve success. This surprising result suggests potentially fruitful directions for the improvement of computational protein design technology.protein engineering | high-throughput stability determination | library design | molecular dynamics | NMR ensemble P rotein-engineering efforts based on directed evolution have met with considerable success (1-3). In tandem, structurebased computational protein design (CPD) methods have been developed to allow screening for desirable sequences to be performed in silico (4-6). Despite a number of high-profile results that demonstrate the utility of CPD (7-12), the routine computational design of functional proteins remains elusive. Thus, many current efforts focus on the improvement of CPD methodology or on the synergistic application of CPD with experimental high-throughput screening or selection (13).Although the stability, solubility, and activity of a protein depend on the relative energetic contributions of many conformational states, including ensembles of native, unfolded, and aggregated structures (14), most CPD methods evaluate sequences based on their energies in the context of one fixed-backbone structure. This simplification has made design results undesirably sensitive to slight changes in main-chain and side-chain conformation and has made difficult the selection of sequences with amino acid composition similar to naturally occurring proteins. These issues have been approached via the use of high-resolution structural templates, expanded rotamer libraries (15, 16), energy functions with softened repulsive terms (10,17,18), iteration between structural refinement and sequence design (10, 19), and amino ac...
Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell‐free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli‐based cell‐free transcription‐translation system. Total yields as high as 1.2 mg mL−1 were observed after a 20‐h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post‐CFPS addition of copper ions in the form of CuSO4. Scale‐up reactions were achieved from 15 to 100 µL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell‐free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.