Protonation State Research Articles

Paramagnetic relaxivity of delocalized long-lived states of protons in chains of CH2 groups.

Long-lived states (LLSs) have lifetimes that can be much longer than longitudinal relaxation times . In molecules containing several geminal pairs of protons in neighboring CH groups, it has been shown that delocalized LLSs can be excited by converting magnetization into imbalances between the populations of singlet and triplet states of each pair. Since the empirical yield of the conversion and reconversion of observable magnetization into LLSs and back is on the order of 10 % if one uses spin-lock induced crossing (SLIC), it would be desirable to boost the sensitivity by dissolution dynamic nuclear polarization (d-DNP). To enhance the magnetization of nuclear spins by d-DNP, the analytes must be mixed with radicals such as 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL). After dissolution, these radicals lead to an undesirable paramagnetic relaxation enhancement (PRE) which shortens not only the longitudinal relaxation times but also the lifetimes of LLSs. It is shown in this work that PRE by TEMPOL is less deleterious for LLSs than for longitudinal magnetization for four different molecules: 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), homotaurine, taurine, and acetylcholine. The relaxivities (i.e., the slopes of the relaxation rate constants as a function of the radical concentration) are 3 to 5 times smaller than the relaxivities of longitudinal magnetization. Partial delocalization of the LLSs across neighboring CH groups may decrease this advantage, but in practice, this effect was observed to be small, for example, when comparing taurine containing two CH groups and homotaurine with three CH groups. Regardless of whether the LLSs are delocalized or not, it is shown that PRE should not be a major problem for experiments combining d-DNP and LLSs, provided the concentration of paramagnetic species after dissolution does not exceed 1 mM, a condition that is readily fulfilled in typical d-DNP experiments. In bullet d-DNP experiments however, it may be necessary to decrease the concentration of TEMPOL or to add ascorbate for chemical reduction.

Theoretical Study on the Copper-Catalyzed ortho-Selective C-H Functionalization of Naphthols with α-Phenyl-α-Diazoesters.

The aromatic C(sp2)-H functionalization of unprotected naphthols with α-phenyl-α-diazoesters under mild conditions catalyzed by CuCl and CuCl2 exhibits high efficiency and unique ortho-selectivity. In this study, the combination of density functional theory (DFT) calculations and experiments is employed to investigate the mechanism of C-H functionalization, which reveals the fundamental origin of the site-selectivity. It explains that CuCl-catalyzed ortho-selective C-H functionlization is due to the bimetallic carbene, which differs from the reaction catalyzed by CuCl2 via monometallic carbene. The results demonstrate the function of favourable H-bond interactions on the site- and chemo-selectivity of reaction through stabilizing the rate-determining transition states in proton (1,3)-migration.

Molecular Mechanism of Spectral Tuning by Chloride Binding in Monkey Green Sensitive Visual Pigment

The visual pigments of the cones perceive red, green, and blue colors. The monkey green (MG) pigment possesses a unique Cl- binding site; however, its relationship to the spectral tuning in green pigments remains elusive. Recently, FTIR spectroscopy revealed the characteristic structural modifications of the retinal binding site by Cl- binding. Herein, we report the computational structural modeling of MG pigments and quantum-chemical simulation to investigate its spectral redshift and physicochemical relevance when Cl- is present. Our protein structures reflect the previously suggested structural changes. AlphaFold2 failed to predict these structural changes. Excited-state calculations successfully reproduced the experimental red-shifted absorption energies, corroborating our protein structures. Electrostatic energy decomposition revealed that the redshift results from the His197 protonation state and conformations of Glu129, Ser202, and Ala308; however, Cl- itself contributes to the blueshift. Site-directed mutagenesis supported our analysis. These modeled structures may provide a valuable foundation for studying cone pigments.

Insight into the Role of Active Site Protonation States and Water Molecules in the Catalytic Inhibition of DPP4 by Vildagliptin.

Vildagliptin (VIL) is an antidiabetic drug that inhibits dipeptidyl peptidase-4 (DPP4) through a covalent mechanism. The molecular bases for this inhibitory process have been addressed experimentally and computationally. Nevertheless, relevant issues remain unknown such as the roles of active site protonation states and conserved water molecules nearby the catalytic center. In this work, molecular dynamics simulations were applied to examine the structures of 12 noncovalent VIL-DPP4 complexes encompassing all possible protonation states of three noncatalytic residues (His126, Asp663, Asp709) that were inconclusively predicted by different computational tools. A catalytically competent complex structure was only achieved in the system with His126 in its ε-form and nonconventional neutral states for Asp663/Asp709. This complex suggested the involvement of one water molecule in the catalytic process of His740/Ser630 activation, which was confirmed by QM/MM simulations. Our findings support the suitability of a novel water-mediated mechanism in which His740/Ser630 activation occurs concertedly with the nucleophilic attack on VIL and the imidate protonation by Tyr547. Then, the restoration of His740/ Tyr547 protonation states occurs via a two-water hydrogen bonding network in a low-barrier process, thus describing the final step of the catalytic cycle for the first time. Additionally, two hydrolytic mechanisms were proposed based on the hydrogen bonding networks formed by water molecules and the catalytic residues along the inhibitory mechanism. These findings are valuable to unveil the molecular features of the covalent inhibition of DPP4 by VIL and support the future development of novel derivatives with improved structural or mechanistic profiles.

Solvation effects on glyphosate protonation and deprotonation states evaluated by mass spectrometry and explicit solvation simulations.

Glyphosate is a widely used herbicide, and its protonation and deprotonation sites are fundamental to understanding its properties. In this work, the sodiated, protonated, and deprotonated glyphosate were evaluated in the gas phase by infrared multiple photon dissociation spectroscopy to determine the exact nature of these coordination, protonation, and deprotonation states in the gas phase. In this context, Natural Bond Orbital analyses were carried out to unravel interactions that govern glyphosate (de)protonation states in the gas phase. The solvent effect on the protonation/deprotonation equilibria was also investigated by implicit (Solvation Model Based on Density and polarizable continuum models) and explicit solvation models (Monte Carlo and Molecular Dynamics simulations). These results show that glyphosate is protonated in the phosphonate group in the gas phase because of the strong hydrogen bond between the carboxylic oxygen (O7) and the protonated phosphonate group (O8-H19), while the most stable species in water is protonated at the amino group because of the preferential interaction of the NH2 + group and the solvent water molecules. Similarly, deprotonated glyphosate [Glyp-H]- was shown to be deprotonated at the phosphonate group in the gas phase but not in solution, also because of the preferential solvation of the NH2 + group present in the other deprotomers. Therefore, these results show that the stabilization of the protonated amino group by the solvent molecules is the governing factor of the (de)protonation equilibrium of glyphosate in water.

Energetic Diversity in the Electron-Transfer Pathways of Type I Photosynthetic Reaction Centers.

Photosynthetic reaction centers from heliobacteria (HbRC) and green sulfur bacteria (GsbRC) are homodimeric proteins and share a common ancestor with photosystem I (PSI), classified as type I reaction centers. Using the HbRC crystal structure, we calculated the redox potential (Em) values in the electron-transfer branches, solving the linear Poisson-Boltzmann equation and considering the protonation states of all titratable sites in the entire protein-pigment complex. Em(A-1) for bacteriochlorophyll g at the secondary site in HbRC (-1157 mV) is as low as Em(A-1) for chlorophyll a in PSI (-1173 mV). Em(A0/HbRC) is at the same level as Em(A0/GsbRC) and is 200 mV higher than Em(A0/PSI) due to the replacement of PsaA-Trp697/PsaB-Trp677 in PSI with PshA-Arg554 in HbRC. In contrast, Em(FX) for the Fe4S4 cluster in HbRC (-420 mV) is significantly higher than Em(FX) in GsbRC (-719 mV) and PSI (-705 mV) due to the absence of acidic residues that correspond to PscA-Asp634 in GsbRC and PsaB-Asp575 in PSI. It seems likely that type I reaction centers have evolved, adopting (bacterio)chlorophylls suitable for their light environments while maintaining electron-transfer cascades.

Torque generation mechanism in Fo motor of ATP synthase elucidated by free-energy and Coulomb-energy landscapes along the c-ring rotation

Fo portion of ATP synthase is a proton-motive rotary motor. The Coulombic attraction between the conserved acidic residues in the c-ring and the arginine in the a-subunit (aR) was early proposed to drive the c-ring rotation relative to the a-subunit, and has been actually observed in our previous molecular dynamics simulation with full atomistic description of Fo embedded in the membrane. In this study, to quantify the driving force, we conducted the umbrella sampling (US) and obtained the free-energy landscape for the c-ring rotation. We first show that the free-energy gradient toward the ATP-synthesis direction appears in the deprotonated state of cE. Using the sampled snapshots that cover a wide range of the rotational angle, we further analyzed the rotational-angle dependence of the hydration and the protonation states and obtained the Coulomb-energy landscapes with a focus on the cE-aR interaction. The results indicate that both the Coulombic solvation energy of cE and the interaction energy between cE and aR contribute to the torque generation for the c-ring rotation.

Selectivity through ion modulated changes in pKa values.

In bacterial voltage gated sodium (Nav) channels, the passage of ions through the pore is controlled by a selectivity filter (SF) comprised of four glutamate residues. The mechanism of selectivity has been the subject of intense research, with suggested mechanisms based on steric effects, and ion triggered conformational change. Here we propose a novel mechanism based on ion-triggered shift in pKa values of SF glutamates. We study the NavMs channel for which the open channel structure is available. Our free energy calculations based on molecular dynamics simulations suggest that pKa values of the four glutamates are higher in solution of K+ ions, than in solution of Na+ ions. Since pKa values are close to the physiological pH, this results in predominant population of the fully deprotonated state of glutamates in Na+ solution, while protonated states are predominantly populated in K+ solution. Through molecular dynamics simulations we also calculate the conductances of the relevant protonation states. The deprotonated state is the most conductive, single protonated state is less conductive, and double protonated state has further reduced conductance. Thus, we propose that the selectivity is achieved through ion triggered shift in protonation state which favors more conductive states for Na+ ions and less conductive states for K+ ions. This mechanism would also suggest pH dependence of selectivity, which was experimentally observed in structurally similar NaChBas channels.

The electronic structure of genome editors from the first principles

Ab-initio molecular dynamics enables following the dynamics of biological systems from the first principles, describing the electronic structure and offering the opportunity to “watch” the evolution of biochemical processes with unique resolution, beyond the capabilities of state-of-the-art experimental techniques. This article reports the role of first-principles (ab-initio) molecular dynamics (MD) in the CRISPR-Cas9 genome editing revolution, achieving a profound understanding of the enzymatic function and offering valuable insights for enzyme engineering. We introduce the methodologies and explain the use of ab-initio MD simulations to establish the two-metal dependent mechanism of DNA cleavage in the RuvC domain of the Cas9 enzyme, and how a second catalytic domain, HNH, cleaves the target DNA with the aid of a single metal ion. A detailed description of how ab-initio MD is combined with free-energy methods—i.e., thermodynamic integration and metadynamics—to break and form chemical bonds is given, explaining the use of these methods to determine the chemical landscape and establish the catalytic mechanism in CRISPR-Cas9. The critical role of classical methods is also discussed, explaining theory and application of constant pH MD simulations, used to accurately predict the catalytic residues’ protonation states. Overall, first-principles methods are shown to unravel the electronic structure and reveal the catalytic mechanism of the Cas9 enzyme, providing valuable insights that can serve for the design of genome editing tools with improved catalytic efficiency or controllable activity.

Membrane composition drives sidechain ionization and assembly of transmembrane protein domains.

Ionization states of amino acid residues play significant roles in membrane protein assembly and function; however, they are difficult to decipher experimentally. The analysis is further complicated by the dearth of data on gradients in polarity, electric potentials, and hydration at the protein-membrane interface. Here we examine how electrostatic interactions, suggested to be essential for T-cell receptor (TCR) membrane assembly, could be manipulated by modifying the membrane composition. Novel pH-sensitive ionizable EPR labels were employed to profile heterogeneous dielectric environment along the transmembrane protein-lipid interfaces. Transmembrane α-helical WALP peptide was used for obtaining the profile of effective pK(a) as a function of membrane depth. It was found that effective pK(a) of membrane-buried sidechains can be significantly shifted by varying the membrane surface charge density. A peptide corresponding to the transmembrane domain of TCRα was labeled with pH-sensitive nitroxide and incorporated into liposomes. EPR of this label reported on the sidechain protonation state, membrane insertion, and association of the helices within the membrane. It was found that an increase in negative change density at the membrane surface alters the protonation state of membrane-buried ionizable sidechain in the transmembrane domain of TCRα. Hyperfine Correlation Spectroscopy (HYSCORE) was used to assess effects of sidechain ionization state on water penetration at membrane-peptide interface while Double Electron-Electron Resonance (DEER) quantified the domain agglomeration. Turning the charge of the sidechain “off” increases tendency of the transmembrane domain to agglomerate - a phenomenon that could be critical for the TCR assembly and its degradation. Overall, EPR of ionizable probes is informative for uncovering mechanisms as to how the changes in local environment along the protein-lipid interface, can result in a “tunable” pKa of the ionizable sidechains and drive the structural changes in membrane proteins.

Characterization of the pH response of disordered tubulin tails.

Microtubules, formed by the polymerization of alpha and beta tubulin heterodimers, play an essential role in cell division and motility. The disordered C-terminal tails of each tubulin monomer are major sites of tubulin regulation and, despite being flexible, appear to regulate microtubule dynamics and tubulin interactions with its binding partners. Across species and isoforms, there is a preponderance of acidic amino acids in the C terminal tails, primarily present in polyglutamate tracts. We postulated that these negatively charged patches may lend the tail sensitivity to various protonation states as might be experienced under cellular stress conditions. Using nuclear magnetic resonance (NMR) spectroscopy, an ideal tool to probe the dynamics and properties of small, disordered regions such as the C-terminal tubulin tails, we probed the pH response of alpha and beta tubulin tail peptides from different species. We used point mutations and truncations within the charged regions to probe the robustness of the pH response of these disordered regions. Our results indicate there is a dynamic and attested effect from changes in pH throughout the C-terminal tail of tubulin monomers.

Constant pH molecular dynamics simulations of membrane proteins.

Environment-driven dynamic protonation of titratable residues in a protein plays a key role in membrane transport and pH response in various biological processes. The dominant protonation state of a titratable amino-acid residue depends on environmental conditions like pH, dielectric environment (ionic strength, proximity to water-membrane interfaces), other amino-acid residues in its vicinity, etc. Constant pH Molecular Dynamics (CpHMD) techniques enable the study of dynamic protonation events in a molecular simulation and can be regarded as an in-silico titration process. In CpHMD techniques, the protonation state of a titratable residue is allowed to propagate between the different ionization states. As the protonation states of different residues change, the charge of the total system fluctuates. In hybrid solvent-based schemes, where the changes in the protonation states are handled using an implicit solvent representation, these charge fluctuations do not pose a problem. However, in case of an explicit-solvent representation, where the electrostatics are handled using a particle mesh ewald (PME) scheme, the varying charge poses a problem in reliable evaluation of energetics and thereby the pKa of the titratable residues. A large aqueous fraction in the simulation box is known to mitigate the charge artifacts. However, compared to soluble proteins, membrane protein simulation systems have a large non-aqueous volume fraction, owing to the lipid membrane. In this study, we compare different CpHMD methodologies for estimation of pKa in membrane proteins. These methods include both an explicit solvent-based CpHMD as well as hybrid solvent-based CpHMD. We present the pKa values of the acidic residues (aspartic acid, glutamic acid) from these simulations. In general, the hybrid solvent-based scheme showed a faster convergence compared to the explicit solvent-based scheme. However, in case of buried residues, the hybrid solvent-based scheme took a longer time to converge.

For every retinylidene a force field: Using machine learning to improve molecular simulations of rhodopsins.

G-protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that perform diverse signaling roles in organisms, including humans. This makes them an attractive target for pharmaceuticals, with 134 different GPCRs being currently targeted by roughly 700 drugs. Here we study the retinylidene cofactor of GPCR rhodopsin that is responsible for initiating phototransduction. There are many distinct retinal forms—isomers, rotamers, and protonation states—that all have unique electronic landscapes. However, current molecular dynamics force field libraries are incomplete in that they ignore these differences and treat each retinylidene form identically. It is unknown what effect this has on current molecular dynamics simulations of retinal proteins. Our work addresses this problem by using machine learning methods to generate unique force fields for each of forty retinylidene forms. This approach improved on traditional parameterization schemes by using Born-Oppenheimer molecular dynamics trajectories to sample the retinylidene energetic landscapes. To validate our methods, the new force fields were then used in all-atom molecular dynamics simulations of the entire rhodopsin-membrane-water system. From these simulations we were able to recover hydrogen relaxation rates that closely matched experimental solid-state deuterium (2H) NMR measurements. Comparison of force field parameters showed a small but—given the highly allosteric nature of rhodopsin—potentially significant difference between retinylidene forms. We expect this work will improve simulations of many different types of rhodopsins, and that it will lead to an increased appreciation of the complexity of the retinylidene energetic landscape. Work supported by US NIH (R01EY026041) and US NSF (CHE 1904125, MCB 1817862) and Ministry of Education and Science of the Russian Federation (Project FSRM-2020-0006).

Comparative dynamics and structural analysis of extremophilic serine proteases from diverse environments.

While life is found in a wide range of environments, many of the chemical functions required for homeostasis and reproduction are conserved. A large subset of these functions are fulfilled by proteins, which themselves have common requirements for both structural stability and chemical function. Organisms living in extreme environments, such as psychrophiles in the arctic ocean or thermophiles around hydrothermal vents, must thus develop adaptations to maintain protein structure and function under widely varying conditions. Here, we consider one ubiquitous enzyme class - the serine proteases - which are found in all known cells. Using genomic data from prokaryotes living under different environmental conditions, we model proteases from the S9, S11, S12, and S13 serine protease families. We predict three-dimensional structures of each enzyme using comparative modeling with in silico maturation to correct for known post-translational modifications, also correcting protonation states for each enzyme's native environment. Using atomistic molecular dynamics (MD) simulations, we obtain locally equilibrated explicit solvent structures for each enzyme, as well as longer trajectories for selected cases drawn from different thermal environments. We perform a comparative analysis of structural properties including surface exposures and secondary structure content across environmental conditions, showing both conserved and variable properties by environment. We also employ protein structure networks to examine structural cohesion, and active site networks to examine differences in active site structure and dynamics. Our analyses suggest patterns of adaptation within the serine proteases, in response to the challenges of life in distinct environmental conditions.

Principles of DNA cleavage in CRISPR-Cas9.

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in double-stranded DNA. In this system, the catalysis of target DNA cleavage by the highly flexible HNH domain is undefined, with two possible conformations whose catalytic relevance has remained unmet. Here, extensive molecular simulations - harnessing multi-microsecond molecular dynamics, quantum mechanics and free energy approaches - are combined with solution NMR and DNA cleavage assays to establish how the HNH nuclease is activated and cleaves the target DNA. We show that the conformation of the active state is critically dependent on the presence of Mg2+, unveiling its cardinal structural role in the organization of the active site. Solution NMR, DNA cleavage assays and molecular simulations of the Mg2+-bound HNH report on the formation of this active state, and consistently show that the protonation state of catalytic H840 is strongly affected by residue mutations within the active site. Finally, quantum mechanical simulations establish that DNA cleavage occurs through the identified active state, showing that the catalysis is activated by H840 and completed by K866, in line with DNA cleavage experiments. Overall, high-level computations, supported by experimental evaluation, resolve the catalytic mechanism and which of the known HNH conformations is responsible for target DNA cleavage in CRISPR-Cas9. This information helps enhancing the catalytic efficiency of Cas9 and its specificity, furthering the development of genome-editing tools.

Residues involved in proton-coupled transport of antimalarial drugs by PfCRT.

A major setback in the fight against malaria has been the emergence of P. falciparum parasites resistant to chloroquine (CQ), the primary antimalarial drug. Neutral CQ enters the parasite's digestive vacuole (DV) and obtains two protons preventing its passive efflux through the membrane. The trapped CQ2+ kills the parasite by disrupting the detoxification process of heme released from proteolyzed host hemoglobin. CQ-resistant parasites show mutations in a DV membrane transporter, P. falciparum CQ resistance transporter (PfCRT), that enables active, proton-coupled efflux of CQ2+. While biochemical measurements established the coupling of PfCRT's transport function to the DV membrane proton gradient, the proton-coupled transport mechanism has remained elusive. Particularly it is still unclear what the proton binding site(s) is(are). Recently, the cryo-EM structure of PfCRT from a CQ-resistant variant was determined with a bound Fab fragment occupying its binding pocket. Using us-long all-atom molecular dynamics (MD) simulations we examined the stability of the transporter in the DV-open conformation in the absence of bound Fab fragments, discovering surprising protein stability upon removal of Fab. Next, utilizing constant pH (cpHMD) simulations at 7 different pH values, and considering Galvani potential offset corrections, we determined the pKa of the four titratable residues (H97, D137, D326, and D329) lining the binding pocket. These simulations allowed unambiguous determination of the primary site responding to DV's acidic environment. Finally, with protonation states compatible with the acidic pH of the DV for the aforementioned residues, we studied binding of CQ2+ to the transporter via a series of driven MD simulations. We then repeated the cpHMD simulations and determined the proton binding site in the presence of the bound CQ2+. Our results shed light on PfCRT's transport mechanism and open new possibilities to design new effective drugs against malaria.

Inferring the transport stoichiometry of the proton/zinc transporter YiiP from microscale thermophoresis and simulations.

The transport stoichiometry of the Zn2+/H+ antiporter YiiP remains an open question. We used experimental microscale thermophoresis (MST) zinc binding data in combination with a microscopic thermodynamic model and constant pH simulation in order to infer the likely microstates of H+ and Zn2+ binding. The pH dependence of Zn2+ binding in different binding sites was measured with MST. For these sites, we constructed microscopic, thermodynamically consistent models based on the pKas of the binding residues and zinc binding affinities. Such a model relates the macroscopically measured MST binding curves to microscopic protonation states and zinc binding in a concentration and pH-dependent manner. We inferred microscopic model parameters from the experimental data with a Monte Carlo approach. We compared the MST-inferred microscopic parameters to results from atomistic constant pH MD simulations in the absence of Zn2+. For zinc binding site B on a solvent-exposed loop between transmembrane helices 2 and 3, MST and simulation data broadly agree with each other. For the transport site A in the transmembrane domain, the CpHMD simulations indicate that the binding site His residue is protonated before any Asp. Using the CpHMD results as a starting point for the MST inference approach, we find that the microstate distribution that best fits the experimental data indicates competitive binding between either one Zn2+ ion or two or three protons bound, with the one-proton state suppressed, possibly due to high cooperativity between the Asp and His residues. The microscopic model indicates a transport stoichiometry of 1:2, or possibly 1:3 at lower pH.

NMR-assisted crystallography of the tryptophan synthase α-aminoacrylate intermediate: Proton positions and role in inhibition.

NMR-assisted crystallography - the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry - holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into structure and chemical dynamics. Here, we make use of this combined approach to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography identifies the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains, and the location and orientation of crystallographic waters within the active site. From this, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the substrate L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate, indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react.

Electronic structure calculations and deuterium NMR spectroscopy reveal retinal mobility in rhodopsin activation.

G-protein coupled receptors (GPCRs) regulate a number of physiological processes, where the visual receptor rhodopsin is an excellent prototype to investigate the structural fluctuations underlying GPCR function. Several X-ray crystal structures are available for the dark and active Meta-II states of rhodopsin. However, because of dehydrating conditions used to crystalize the protein, the conformation and orientation of the retinal chromophore in its binding pocket remain uncertain. Our hypothesis was that greater retinal mobility is needed in the active Meta-II state for the interaction with effector proteins. Accordingly, we investigated the structure and orientation of retinal in the dark and the Meta-II states using density functional theory (DFT) calculations and deuterium NMR (2H NMR) spectroscopy. For the computational simulations, we modeled the protein environment by including a number of amino acid residues in the retinal binding pocket and the water molecules around it. These models were tested with different chromophore orientations and protonation states of Glu113 and Glu181. Next, we performed DFT and complete active space (CAS) calculations to characterize the electronic structure of the dark and Meta-II states of rhodopsin. 2H NMR spectroscopic studies were also conducted on the active Meta-II state that contained deuterium-labelled retinal to experimentally validate the possible retinal orientations. The experiment revealed two possible retinal orientations (“flipped” and “unflipped”) without significant steric clashes in the binding pocket of active Meta-II state. By contrast, the DFT calculations show the Meta-II state predominantly contains the “flipped” orientation while the dark-state predominantly accommodates the “unflipped” retinal orientation. Thus the chromophore of the visual receptor undergoes significant mobility changes upon light activation of rhodopsin. [1] A.V. Struts et al. (2011) Nat.Struct.Mol.Biol. 18, 392–394, [2] M.N. Ryazantsev et al. (2019) J.Membr.Biol. 252, 425–449.

Calcium-dependent membrane interactions of tumor-targeting peptide pHLIP.

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pHLIP (pH-Low Insertion Peptide). Here, we explore the effects of PS, Ca2+, and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond-timescale all-atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide-lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH-induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+-induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to link cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+-dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid-coupled regulation of protein-membrane insertion by divalent cations.