Electric Fields and Enzyme Catalysis.

What happens inside an enzyme's active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists' attention for a long time. Computer models of increasing sophistication have predicted an important role for electrostatic interactions in enzymatic reactions, yet this hypothesis has proved vexingly difficult to test experimentally. Recent experiments utilizing the vibrational Stark effect make it possible to measure the electric field a substrate molecule experiences when bound inside its enzyme's active site. These experiments have provided compelling evidence supporting a major electrostatic contribution to enzymatic catalysis. Here, we review these results and develop a simple model for electrostatic catalysis that enables us to incorporate disparate concepts introduced by many investigators to describe how enzymes work into a more unified framework stressing the importance of electric fields at the active site.

Membrane localization accelerates association under conditions relevant to cellular signaling.

Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here, we directly compare an identical association reaction, the binding of complementary DNA strands, in solution and on supported membranes. The measured rate constants show that for a 10-µm-radius spherical cell, association is 22- to 33-fold faster at the membrane than in the cytoplasm. The kinetic advantage depends on cell size and is essentially negligible for typical ~1 µm prokaryotic cells. The rate enhancement is attributable to a combination of higher encounter rates in two dimensions and a higher reaction probability per encounter.

Critical Evaluation of Polarizable and Nonpolarizable Force Fields for Proteins Using Experimentally Derived Nitrile Electric Fields.

Molecular dynamics (MD) simulations are frequently carried out for proteins to investigate the role of electrostatics in their biological function. The choice of force field (FF) can significantly alter the MD results, as the simulated local electrostatic interactions lack benchmarking in the absence of appropriate experimental methods. We recently reported that the transition dipole moment (TDM) of the popular nitrile vibrational probe varies linearly with the environmental electric field, overcoming well-known hydrogen bonding (H-bonding) issues for the nitrile frequency and, thus, enabling the unambiguous measurement of electric fields in proteins (J. Am. Chem. Soc. 2022, 144 (17), 7562-7567). Herein, we utilize this new strategy to enable comparisons of experimental and simulated electric fields in protein environments. Specifically, previously determined TDM electric fields exerted onto nitrile-containing o-cyanophenylalanine residues in photoactive yellow protein are compared with MD electric fields from the fixed-charge AMBER FF and the polarizable AMOEBA FF. We observe that the electric field distributions for H-bonding nitriles are substantially affected by the choice of FF. As such, AMBER underestimates electric fields for nitriles experiencing moderate field strengths; in contrast, AMOEBA robustly recapitulates the TDM electric fields. The FF dependence of the electric fields can be partly explained by the presence of additional negative charge density along the nitrile bond axis in AMOEBA, which is due to the inclusion of higher-order multipole parameters; this, in turn, begets more head-on nitrile H-bonds. We conclude by discussing the implications of the FF dependence for the simulation of nitriles and proteins in general.

Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the ∼4-ps population, P+HA- forms through a two-step process, P*→ P+BA-→ P+HA-, while in the ∼20-ps population, it forms via a one-step P* → P+HA- superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA- intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P+BA- along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA- formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration by using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display standard deviations in composition (n = 30 GUVs) on the order of 1-4 mol %, consistent with variability seen in previous indirect measurements.

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ß-barrel across the time window of our measurements.

Membrane-tethered mucin-like polypeptides sterically inhibit binding and slow fusion kinetics of influenza A virus.

The mechanism(s) by which cell-tethered mucins modulate infection by influenza A viruses (IAVs) remain an open question. Mucins form both a protective barrier that can block virus binding and recruit IAVs to bind cells via the sialic acids of cell-tethered mucins. To elucidate the molecular role of mucins in flu pathogenesis, we constructed a synthetic glycocalyx to investigate membrane-tethered mucins in the context of IAV binding and fusion. We designed and synthesized lipid-tethered glycopolypeptide mimics of mucins and added them to lipid bilayers, allowing chemical control of length, glycosylation, and surface density of a model glycocalyx. We observed that the mucin mimics undergo a conformational change at high surface densities from a compact to an extended architecture. At high surface densities, asialo mucin mimics inhibited IAV binding to underlying glycolipid receptors, and this density correlated to the mucin mimic's conformational transition. Using a single virus fusion assay, we observed that while fusion of virions bound to vesicles coated with sialylated mucin mimics was possible, the kinetics of fusion was slowed in a mucin density-dependent manner. These data provide a molecular model for a protective mechanism by mucins in IAV infection, and therefore this synthetic glycocalyx provides a useful reductionist model for studying the complex interface of host-pathogen interactions.

Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.

The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.

ß-Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields.

A compound bound covalently to an enzyme active site can act either as a substrate if the covalent linkage is readily broken up by the enzyme or as an inhibitor if the bond dissociates slowly. We tracked the reactivity of such bonds associated with the rise of the resistance to penicillin G (PenG) in protein evolution from penicillin-binding proteins (PBPs) to TEM ß-lactamases and with the development of avibactam (Avb) to overcome the resistance. We found that the ester linkage in PBP-PenG is resistant to hydrolysis mainly due to the small electric fields present in the protein active site. Conversely, the same linkage in the descendant TEM-PenG experiences large electric fields that stabilize the more charge-separated transition state and thus lower the free energy barrier to hydrolysis. Specifically, the electric fields were improved from -59 to -140 MV/cm in an ancient evolution dating back billions of years, contributing 5 orders of magnitude rate acceleration. This trend continues today in the nullification of newly developed antibiotic drugs. The fast linkage hydrolysis acquired from evolution is counteracted by the upgrade of PenG to Avb whose linkage escapes from the hydrolysis by returning to a low-field environment. Using the framework of electrostatic catalysis, the electric field, an observable from vibrational spectroscopy, provides a unifying physical metric to understand protein evolution and to guide the design of covalent drugs.

Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments.

Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the electric field projected on the -C≡N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to both H-bonding and non-H-bonding interactions. It can therefore be generally applied to determine electric fields in all environments. Additionally, it allows for semiempirical extraction of the H-bonding contribution to the blueshift of the nitrile frequency. Nitriles were incorporated at H-bonding and non-H-bonding protein sites using amber suppression, and each nitrile variant was structurally characterized at high resolution. We exploited the combined information available from variations in frequency and integrated intensity and demonstrate that nitriles are a generally useful probe for electric fields.

Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme.

The past decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or nonbiological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most redesigned proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an "excited-state enzyme". Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.

Protein Electric Fields Enable Faster and Longer-Lasting Covalent Inhibition of ß-Lactamases.

The widespread design of covalent drugs has focused on crafting reactive groups of proper electrophilicity and positioning toward targeted amino-acid nucleophiles. We found that environmental electric fields projected onto a reactive chemical bond, an overlooked design element, play essential roles in the covalent inhibition of TEM-1 ß-lactamase by avibactam. Using the vibrational Stark effect, the magnitudes of the electric fields that are exerted by TEM active sites onto avibactam's reactive C═O were measured and demonstrate an electrostatic gating effect that promotes bond formation yet relatively suppresses the reverse dissociation. These results suggest new principles of covalent drug design and off-target site prediction. Unlike shape and electrostatic complementary which address binding constants, electrostatic catalysis drives reaction rates, essential for covalent inhibition, and deepens our understanding of chemical reactivity, selectivity, and stability in complex systems.

Recombination between 13C and 2H to Form Acetylide (13C22H-) Probes Nanoscale Interactions in Lipid Bilayers via Dynamic Secondary Ion Mass Spectrometry: Cholesterol and GM1 Clustering.

Although it is thought that there is lateral heterogeneity of lipid and protein components within biological membranes, probing this heterogeneity has proven challenging. The difficulty in such experiments is due to both the small length scale over which such heterogeneity can occur, and the significant perturbation resulting from fluorescent or spin labeling on the delicate interactions within bilayers. Atomic recombination during dynamic nanoscale secondary ion imaging mass spectrometry (NanoSIMS) is a non-perturbative method for examining nanoscale bilayer interactions. Atomic recombination is a variation on conventional NanoSIMS imaging, whereby an isotope on one molecule combines with a different isotope on another molecule during the ionization process, forming an isotopically enriched polyatomic ion in a distance-dependent manner. We show that the recombinant ion, 13C22H-, is formed in high yield from 13C- and 2H-labeled lipids. The low natural abundance of triply labeled acetylide also makes it an ideal ion to probe GM1 clusters in model membranes and the effects of cholesterol on lipid-lipid interactions. We find evidence supporting the cholesterol condensation effect as well as the presence of nanoscale GM1 clusters in model membranes.

Modulating the Influenza A Virus-Target Membrane Fusion Interface With Synthetic DNA-Lipid Receptors.

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell-surface glycans are highly heterogeneous in length and glycosylation density, which leads to variations in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion or the low-pH-triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

Single-virus content-mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency.

To infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay in which content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content-loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content-mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage after pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content-mixing kinetics of other enveloped viruses.

Ladderane phospholipids form a densely packed membrane with normal hydrazine and anomalously low proton/hydroxide permeability.

Ladderane lipids are unique to anaerobic ammonium-oxidizing (anammox) bacteria and are enriched in the membrane of the anammoxosome, an organelle thought to compartmentalize the anammox process, which involves the toxic intermediate hydrazine (N2H4). Due to the slow growth rate of anammox bacteria and difficulty of isolating pure ladderane lipids, experimental evidence of the biological function of ladderanes is lacking. We have synthesized two natural and one unnatural ladderane phosphatidylcholine lipids and compared their thermotropic properties in self-assembled bilayers to distinguish between [3]- and [5]-ladderane function. We developed a hydrazine transmembrane diffusion assay using a water-soluble derivative of a hydrazine sensor and determined that ladderane membranes are as permeable to hydrazine as straight-chain lipid bilayers. However, pH equilibration across ladderane membranes occurs 5-10 times more slowly than across straight-chain lipid membranes. Langmuir monolayer analysis and the rates of fluorescence recovery after photobleaching suggest that dense ladderane packing may preclude formation of proton/hydroxide-conducting water wires. These data support the hypothesis that ladderanes prevent the breakdown of the proton motive force rather than blocking hydrazine transmembrane diffusion in anammox bacteria.

Enantioselective Total Synthesis of the Archaeal Lipid Parallel GDGT-0 (Isocaldarchaeol)*.

Archaeal glycerol dibiphytanyl glycerol tetraethers (GDGT) are some of the most unusual membrane lipids identified in nature. These amphiphiles are the major constituents of the membranes of numerous Archaea, some of which are extremophilic organisms. Due to their unique structures, there has been significant interest in studying both the biophysical properties and the biosynthesis of these molecules. However, these studies have thus far been hampered by limited access to chemically pure samples. Herein, we report a concise and stereoselective synthesis of the archaeal tetraether lipid parallel GDGT-0 and the synthesis and self-assembly of derivatives bearing different polar groups.

Target Membrane Cholesterol Modulates Single Influenza Virus Membrane Fusion Efficiency but Not Rate.

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, whereas the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature.

A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase.

Electrostatic interactions play a pivotal role in enzymatic catalysis and are increasingly modeled explicitly in computational enzyme design; nevertheless, they are challenging to measure experimentally. Using vibrational Stark effect (VSE) spectroscopy, we have measured electric fields inside the active site of the enzyme ketosteroid isomerase (KSI). These studies have shown that these fields can be unusually large, but it has been unclear to what extent they specifically stabilize the transition state (TS) relative to a ground state (GS). In the following, we use crystallography and computational modeling to show that KSI's intrinsic electric field is nearly perfectly oriented to stabilize the geometry of its reaction's TS. Moreover, we find that this electric field adjusts the orientation of its substrate in the ground state so that the substrate needs to only undergo minimal structural changes upon activation to its TS. This work provides evidence that the active site electric field in KSI is preorganized to facilitate catalysis and provides a template for how electrostatic preorganization can be measured in enzymatic systems.

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore.

The neutral or A state of the green fluorescent protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the protein environment and accounts for the large Stokes shift of GFP in response to near UV excitation. Its color tuning mechanism has been largely overlooked, as it is less preferred for imaging applications than the redder anionic or B state. Past studies, based on site-directed mutagenesis or solvatochromism of the isolated chromophore, have concluded that its color tuning range is much narrower than its anionic counterpart. However, as we performed extensive investigation on more GFP mutants, we found that the color of the neutral chromophore can be more sensitive to protein electrostatics than can the anionic counterpart. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model as compared to that of the anionic state, which requires only two forms ( J. Am. Chem. Soc. 2019, 141, 15250-15265). Specifically, an underlying zwitterionic charge-transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to excited-state proton transfer (ESPT) of the protonated chromophore, we infer design principles of the GFP chromophore as a photoacid through the color tuning mechanisms of both protonation states. The three-form model could also be applied to similar biological and nonbiological dyes and complements the failure of the two-form model for donor-acceptor systems with localized ground-state electronic distributions.