Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization.

Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p-chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔTm' = +27 °C) and temperature-induced heme loss (ΔT50 = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first 6 months of the COVID-19 pandemic.

Understanding the molecular evolution of the SARS-CoV-2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three-dimensional structures of SARS-CoV-2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes. Catalytic residues in active sites and binding residues in protein-protein interfaces showed modest, but significant, numbers of substitutions, highlighting the mutational robustness of the viral proteome. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for potential drug discovery targets and the four structural proteins that comprise the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and protein-protein and protein-nucleic acid interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Enantioselective Synthesis of α-Trifluoromethyl Amines via Biocatalytic N-H Bond Insertion with Acceptor-Acceptor Carbene Donors.

The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N-H carbene insertion reaction catalyzed by engineered variants of cytochrome c552 from Hydrogenobacter thermophilus. Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched ß-trifluoromethyl-ß-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N-H carbenoid insertion with an acceptor-acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.

Flexible boundary layer using exchange for embedding theories. I. Theory and implementation.

Embedding theory is a powerful computational chemistry approach to exploring the electronic structure and dynamics of complex systems, with Quantum Mechanical/Molecular Mechanics (QM/MM) being the prime example. A challenge arises when trying to apply embedding methodology to systems with diffusible particles, e.g., solvents, if some of them must be included in the QM region, for example, in the description of solvent-supported electronic states or reactions involving proton transfer or charge-transfer-to-solvent: without a special treatment, inter-diffusion of QM and MM particles will eventually lead to a loss of QM/MM separation. We have developed a new method called Flexible Boundary Layer using Exchange (FlexiBLE) that solves the problem by adding a biasing potential to the system that closely maintains QM/MM separation. The method rigorously preserves ensemble averages by leveraging their invariance to an exchange of identical particles. With a careful choice of the biasing potential and the use of a tree algorithm to include only important QM and MM exchanges, we find that the method has an MM-forcefield-like computational cost and thus adds negligible overhead to a QM/MM simulation. Furthermore, we show that molecular dynamics with the FlexiBLE bias conserves total energy, and remarkably, sub-diffusional dynamical quantities in the inner QM region are unaffected by the applied bias. FlexiBLE thus widens the range of chemistry that can be studied with embedding theory.

Flexible boundary layer using exchange for embedding theories. II. QM/MM dynamics of the hydrated electron.

The FlexiBLE embedding method introduced in Paper I [Z. Shen and W. J. Glover, J. Chem. Phys. 155, 224112 (2021)] is applied to explore the structure and dynamics of the aqueous solvated electron at an all-electron density functional theory Quantum Mechanics/Molecular Mechanics level. Compared to a one-electron mixed quantum/classical description, we find the dynamics of the many-electron model of the hydrated electron exhibits enhanced coupling to water OH stretch modes. Natural bond orbital analysis reveals this coupling is due to significant population of water OH σ* orbitals, reaching 20%. Based on this, we develop a minimal frontier orbital picture of the hydrated electron involving a cavity orbital and important coupling to 4-5 coordinating OH σ* orbitals. Implications for the interpretation of the spectroscopy of this interesting species are discussed.

Highly stereoselective and enantiodivergent synthesis of cyclopropylphosphonates with engineered carbene transferases.

Organophosphonate compounds have represented a rich source of biologically active compounds, including enzyme inhibitors, antibiotics, and antimalarial agents. Here, we report the development of a highly stereoselective strategy for olefin cyclopropanation in the presence of a phosphonyl diazo reagent as carbene precursor. In combination with a 'substrate walking' protein engineering strategy, two sets of efficient and enantiodivergent myoglobin-based biocatalysts were developed for the synthesis of both (1R,2S) and (1S,2R) enantiomeric forms of the desired cyclopropylphosphonate ester products. This methodology enables the efficient transformation of a broad range of vinylarene substrates at a preparative scale (i.e. gram scale) with up to 99% de and ee. Mechanistic studies provide insights into factors that contribute to make this reaction inherently more challenging than hemoprotein-catalyzed olefin cyclopropanation with ethyl diazoacetate investigated previously. This work expands the range of synthetically useful, enzyme-catalyzed transformations and paves the way to the development of metalloprotein catalysts for abiological carbene transfer reactions involving non-canonical carbene donor reagents.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic.

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.