RESUMO
Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound holo-conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.
RESUMO
The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (µs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) "locked off", Fe(II)-CO "on", and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.
Assuntos
Proteínas de Bactérias , Monóxido de Carbono , Espectroscopia de Ressonância de Spin Eletrônica , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Heme/química , Heme/metabolismo , Ligação Proteica , Conformação Proteica , Regulação Alostérica , Hemeproteínas , TransativadoresRESUMO
Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.
Assuntos
Calmodulina , Espectrofotometria Infravermelho , Calmodulina/química , Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Ligação Proteica , Simulação de Acoplamento MolecularRESUMO
Blue copper proteins are models for illustrating how proteins tune metal properties. Nevertheless, the mechanisms by which the protein controls the metal site remain to be fully elucidated. A hindrance is that the closed shell Cu(I) site is inaccessible to most spectroscopic analyses. Carbon deuterium (C-D) bonds used as vibrational probes afford nonperturbative, selective characterization of the key cysteine and methionine copper ligands in both redox states. The structural integrity of Nostoc plastocyanin was perturbed by disrupting potential hydrogen bonds between loops of the cupredoxin fold via mutagenesis (S9A, N33A, N34A), variably raising the midpoint potential. The C-D vibrations show little change to suggest substantial alteration to the Cu(II) coordination in the oxidized state or in the Cu(I) interaction with the cysteine ligand. They rather indicate, along with visible and NMR spectroscopy, that the methionine ligand distinctly interacts more strongly with the Cu(I) ion, in line with the increases in midpoint potential. Here we show that the protein structure determines the redox properties by restricting the interaction between the methionine ligand and Cu(I) in the reduced state.
RESUMO
Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3-, H3C-, and iPr-). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.
Assuntos
Metaloproteínas , Metaloproteínas/química , Cristalografia por Raios X , Escherichia coli/metabolismo , Dicroísmo Circular , CinéticaRESUMO
Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN-heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.
Assuntos
Mononucleotídeo de Flavina , Heme , Domínio Catalítico , Transporte de Elétrons , Mononucleotídeo de Flavina/química , Heme/química , Heme Oxigenase (Desciclizante) , Humanos , Ligantes , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo II/químicaRESUMO
Proteins tune the reactivity of metal sites; less understood is the impact of association with a redox partner. We demonstrate the utility of carbon-deuterium labels for selective analysis of delicate metal sites. Introduced into plastocyanin, they reveal substantial strengthening of the key Cu-Cys89 bond upon association with cytochrome f.
Assuntos
Cobre , Plastocianina , Carbono , Cobre/química , Citocromos f/metabolismo , Deutério , Oxirredução , Plastocianina/química , Plastocianina/metabolismoRESUMO
Proteins are complex, heterogeneous macromolecules that exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires experimental tools to characterize them with high spatial and temporal precision. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution. Two-dimensional (2D) IR methods that provide richer information are becoming more routine but remain challenging to apply to proteins. Spectral congestion typically prevents selective investigation of native vibrations; however, the problem can be overcome by site-specific introduction of amino acid side chains that have vibrational groups with frequencies in the "transparent window" of protein spectra. This Perspective provides an overview of the history and recent progress in the development of transparent window 2D IR of proteins.
Assuntos
Proteínas/química , Espectrofotometria Infravermelho/métodos , Monóxido de Carbono/química , Cianetos/química , Ligantes , Metais/química , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Proteins function as ensembles of interconverting structures. The motions span from picosecond bond rotations to millisecond and longer subunit displacements. Characterization of functional dynamics on all spatial and temporal scales remains challenging experimentally. Two-dimensional infrared spectroscopy (2D IR) is maturing as a powerful approach for investigating proteins and their dynamics. We outline the advantages of IR spectroscopy, describe 2D IR and the information it provides, and introduce vibrational groups for protein analysis. We highlight example studies that illustrate the power and versatility of 2D IR for characterizing protein dynamics and conclude with a brief discussion of the outlook for biomolecular 2D IR.
Assuntos
Proteínas , Espectrofotometria InfravermelhoRESUMO
Cytochrome P450s are diverse and powerful catalysts that can activate molecular oxygen to oxidize a wide variety of substrates. Catalysis relies on effective uptake of two electrons and two protons. For cytochrome P450cam, an archetypal member of the superfamily, the second electron must be supplied by the redox partner putidaredoxin (Pdx). Pdx also plays an effector role beyond electron transfer, but after decades the mechanism remains under investigation. We applied infrared spectroscopy to heme-ligated CN- to examine the influence of Pdx binding. The results indicate that Pdx induces the population of a conformation wherein the CN- ligand forms a strong hydrogen bond to a solvent water molecule, experimentally corroborating the formation of a proposed proton delivery network. Further, characterization of T252A P450cam implicates the side chain of Thr252 in regulating the population equilibrium of hydrogen-bonded states within the P450cam/Pdx complex, which could underlie its role in directing activated oxygen toward product formation and preventing reaction uncoupling through peroxide release.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ferredoxinas/farmacologia , Proteínas de Bactérias/química , Cânfora/química , Cânfora 5-Mono-Oxigenase/química , Catálise , Domínio Catalítico , Cristalografia por Raios X/métodos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Transporte de Elétrons , Ferredoxinas/metabolismo , Heme/química , Ligação de Hidrogênio/efeitos dos fármacos , Cinética , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação ProteicaRESUMO
Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.
Assuntos
Cânfora 5-Mono-Oxigenase , Sistema Enzimático do Citocromo P-450 , Cânfora , Cânfora 5-Mono-Oxigenase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Conformação Proteica , Especificidade por SubstratoRESUMO
Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and interprotein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an all-encompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semiempirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository Web site (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-the-art multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.
Assuntos
Modelos Químicos , Proteínas/química , Análise Espectral/métodos , Humanos , Análise Espectral Raman , Eletricidade Estática , VibraçãoRESUMO
Flexible protein sequences populate ensembles of rapidly interconverting states differentiated by small-scale fluctuations; however, elucidating whether and how the ensembles determine function experimentally is challenged by the combined high spatial and temporal resolution needed to capture the states. We used carbon-deuterium (C-D) bond vibrations incorporated as infrared probes to characterize with residue-specific detail the heterogeneity of states adopted by proline-rich (PR) sequences and assess their involvement in recognition of Src homology 3 domains. The C-D absorption envelopes provided evidence for two or three sub-populations at all proline residues. The changes in the subpopulations induced by binding generally reflected recognition by conformational selection but depended on the residue and the state of the ligand to illuminate distinct mechanisms among the PR ligands. Notably, the spectral data indicate that greater adaptability among the states is associated with reduced recognition specificity and that perturbation to the ensemble populations contributes to differences in binding entropy. Broadly, the study quantifies rapidly interconverting ensembles with residue-specific detail and implicates them in function.
Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Proteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Entropia , Cinética , Peptídeos/química , Peptídeos/metabolismo , Domínios de Homologia de srcRESUMO
Proteins exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires knowledge of the populated states and thus the experimental tools to characterize them. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution, and 2D IR methods that provide richer information are becoming more routine. Although application of IR spectroscopy for investigation of proteins is challenged by spectral congestion, the issue can be overcome by site-specific introduction of amino acid side chains that have IR probe groups with frequency-resolved absorptions, which furthermore enables selective characterization of different locations in proteins. Here, we briefly introduce the biophysical methods and summarize the current progress toward the study of proteins. We then describe our efforts to apply site-specific 1D and 2D IR spectroscopy toward elucidation of protein conformations and dynamics to investigate their involvement in protein molecular recognition, in particular mediated by dynamic complexes: plastocyanin and its binding partner cytochrome f, cytochrome P450s and substrates or redox partners, and Src homology 3 domains and proline-rich peptide motifs. We highlight the advantages of frequency-resolved probes to characterize specific, local sites in proteins and uncover variation among different locations, as well as the advantage of the fast time scale of IR spectroscopy to detect rapidly interconverting states. In addition, we illustrate the greater insight provided by 2D methods and discuss potential routes for further advancement of the field of biomolecular 2D IR spectroscopy.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Citocromos f/química , Simulação de Dinâmica Molecular , Plastocianina/química , Modelos Moleculares , Conformação Proteica , Espectrofotometria InfravermelhoRESUMO
Transient protein complexes are crucial for sustaining dynamic cellular processes. The complexes of electron-transfer proteins are a notable example, such as those formed by plastocyanin (Pc) and cytochrome f (cyt f) in the photosynthetic apparatus. The dynamic and heterogeneous nature of these complexes, however, makes their study challenging. To better elucidate the complex of Nostoc Pc and cyt f, 2D-IR spectroscopy coupled to site-specific labeling with cyanophenylalanine infrared (IR) probes was employed to characterize how the local environments at sites along the surface of Pc were impacted by cyt f binding. The results indicate that Pc most substantially engages with cyt f via the hydrophobic patch around the copper redox site. Complexation with cyt f led to an increase in inhomogeneous broadening of the probe absorptions, reflective of increased heterogeneity of interactions with their environment. Notably, most of the underlying states interconverted very rapidly (1 to 2 ps), suggesting a complex with a highly mobile interface. The data support a model of the complex consisting of a large population of an encounter complex. Additionally, the study demonstrates the application of 2D-IR spectroscopy with site-specifically introduced probes to reveal new quantitative insight about dynamic biochemical systems.
Assuntos
Citocromos f/metabolismo , Plastocianina/metabolismo , Alanina/análogos & derivados , Alanina/química , Sítios de Ligação , Citocromos f/química , Interações Hidrofóbicas e Hidrofílicas , Sondas Moleculares/química , Nitrilas/química , Nostoc/química , Plastocianina/química , Ligação Proteica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The conformational heterogeneity and dynamics of protein side chains contribute to function, but investigating exactly how is hindered by experimental challenges arising from the fast timescales involved and the spatial heterogeneity of protein structures. The potential of two-dimensional infrared (2D IR) spectroscopy for measuring conformational heterogeneity and dynamics with unprecedented spatial and temporal resolution has motivated extensive effort to develop amino acids with functional groups that have frequency-resolved absorptions to serve as probes of their protein microenvironments. We demonstrate the full advantage of the approach by selective incorporation of the probe p-cyanophenylalanine at six distinct sites in a Src homology 3 domain and the application of 2D IR spectroscopy to site-specifically characterize heterogeneity and dynamics and their contribution to cognate ligand binding. The approach revealed a wide range of microenvironments and distinct responses to ligand binding, including at the three adjacent, conserved aromatic residues that form the recognition surface of the protein. Molecular dynamics simulations performed for all the labeled proteins provide insight into the underlying heterogeneity and dynamics. Similar application of 2D IR spectroscopy and site-selective probe incorporation will allow for the characterization of heterogeneity and dynamics of other proteins, how heterogeneity and dynamics are affected by solvation and local structure, and how they might contribute to biological function.
Assuntos
Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Proteínas/química , Espectrofotometria Infravermelho , Simulação de Dinâmica Molecular , Domínios de Homologia de srcRESUMO
Conformational heterogeneity is critical to understanding protein function but challenging to quantify. Experimental approaches that can provide sufficient temporal and spatial resolution to measure even rapidly interconverting states at specific locations in proteins are needed to fully elucidate the contribution of conformational heterogeneity and dynamics to function. Infrared spectroscopy in combination with the introduction of carbon deuterium bonds, which provide frequency-resolved probes of their environments, can uncover rapidly interconverting states with residue-specific detail. Using this approach, we quantify conformational heterogeneity of proline-rich peptides associated with different proline backbone conformations, as well as reveal their dependence on the sequence context.
Assuntos
Peptídeos/química , Prolina/química , Espectrofotometria Infravermelho , Sequência de Aminoácidos , Simulação de Dinâmica Molecular , Estrutura Secundária de ProteínaRESUMO
The importance of conformational dynamics to protein function is now well-appreciated. An outstanding question is whether they are involved in the effector role played by putidaredoxin (Pdx) in its reduction of the O2 complex of cytochrome P450cam (P450cam), an archetypical member of the cytochrome P450 superfamily. Recent studies have reported that binding of Pdx induces a conformational change from a closed to an open state of ferric P450cam, but a similar conformational change does not appear to occur for the ferrous, CO-ligated enzyme. To better understand the effector role of Pdx when binding the ferrous, CO-ligated P450cam, we applied 2D IR spectroscopy to compare the conformations and dynamics of the wild-type (wt) enzyme in the absence and presence of Pdx, as well as of L358P P450cam (L358P), which has served as a putative model for the Pdx complex. The CO vibrations of the Pdx complex and L358P report population of two conformational states in which the CO experiences distinct environments. The dynamics among the CO frequencies indicate that the energy landscape of substates within one conformation are reflective of the closed state of P450cam, and for the other conformation, differ from the free wt enzyme, but are equivalent between the Pdx complex and L358P. The two states co-populated by the Pdx complex are postulated to reflect a loosely bound encounter complex and a more tightly bound state, as is commonly observed for the dynamic complexes of redox partners. Significantly, this study shows that the binding of Pdx to ferrous, CO-ligated P450cam does perturb the conformational ensemble in a way that might underlie the effector role of Pdx.