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1.
Sci Rep ; 8(1): 14745, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30283151

RESUMO

Photosystem II (PSII) reaction centre D1 protein of oxygenic phototrophs is pivotal for sustaining photosynthesis. Also, it is targeted by herbicides and herbicide-resistant weeds harbour single amino acid substitutions in D1. Conservation of D1 primary structure is seminal in the photosynthetic performance in many diverse species. In this study, we analysed built-in and environmentally-induced (high temperature and high photon fluency - HT/HL) phenotypes of two D1 mutants of Chlamydomonas reinhardtii with Ala250Arg (A250R) and Ser264Lys (S264K) substitutions. Both mutations differentially affected efficiency of electron transport and oxygen production. In addition, targeted metabolomics revealed that the mutants undergo specific differences in primary and secondary metabolism, namely, amino acids, organic acids, pigments, NAD, xanthophylls and carotenes. Levels of lutein, ß-carotene and zeaxanthin were in sync with their corresponding gene transcripts in response to HT/HL stress treatment in the parental (IL) and A250R strains. D1 structure analysis indicated that, among other effects, remodelling of H-bond network at the QB site might underpin the observed phenotypes. Thus, the D1 protein, in addition to being pivotal for efficient photosynthesis, may have a moonlighting role in rewiring of specific metabolic pathways, possibly involving retrograde signalling.


Assuntos
Chlamydomonas reinhardtii/genética , Transdução de Sinal Luminoso/genética , Fótons , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/química , Substituição de Aminoácidos , Aminoácidos/metabolismo , Carotenoides/biossíntese , Reprogramação Celular , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Ácidos Dicarboxílicos/metabolismo , Transporte de Elétrons/efeitos da radiação , Expressão Gênica , Temperatura Alta , Ligações de Hidrogênio , Redes e Vias Metabólicas/genética , Modelos Moleculares , Mutação , NAD/metabolismo , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Pigmentos Biológicos/biossíntese , Estrutura Secundária de Proteína , Xantofilas/biossíntese
2.
J Phys Chem B ; 121(29): 6981-6988, 2017 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-28498662

RESUMO

Redox reactions play key roles in fundamental biological processes. The related spatial organization of donors and acceptors is assumed to undergo evolutionary optimization facilitating charge mobilization within the relevant biological context. Experimental information from submolecular functional sites is needed to understand the organization strategies and driving forces involved in the self-development of structure-function relationships. Here we exploit chemically resolved electrical measurements (CREM) to probe the atom-specific electrostatic potentials (ESPs) in artificial arrays of bacteriochlorophyll (BChl) derivatives that provide model systems for photoexcited (hot) electron donation and withdrawal. On the basis of computations we show that native BChl's in the photosynthetic reaction center (RC) self-assemble at their ground-state as aligned gates for functional charge transfer. The combined computational and experimental results further reveal how site-specific polarizability perpendicular to the molecular plane enhances the hot-electron transport. Maximal transport efficiency is predicted for a specific, ∼5 Å, distance above the center of the metalized BChl, which is in remarkably close agreement with the distance and mutual orientation of corresponding native cofactors. These findings provide new metrics and guidelines for analysis of biological redox centers and for designing charge mobilizing machines such as artificial photosynthesis.


Assuntos
Elétrons , Proteínas/química , Simulação por Computador , Espectrometria de Massas , Oxirredução , Silício/química
3.
Sci Rep ; 7: 44580, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28300167

RESUMO

Interquinone QA- → QB electron-transfer (ET) in isolated photosystem II reaction centers (PSII-RC) is protein-gated. The temperature-dependent gating frequency "k" is described by the Eyring equation till levelling off at T ≥ 240 °K. Although central to photosynthesis, the gating mechanism has not been resolved and due to experimental limitations, could not be explored in vivo. Here we mimic the temperature dependency of "k" by enlarging VD1-208, the volume of a single residue at the crossing point of the D1 and D2 PSII-RC subunits in Synechocystis 6803 whole cells. By controlling the interactions of the D1/D2 subunits, VD1-208 (or 1/T) determines the frequency of attaining an ET-active conformation. Decelerated ET, impaired photosynthesis, D1 repair rate and overall cell physiology upon increasing VD1-208 to above 130 Å3, rationalize the >99% conservation of small residues at D1-208 and its homologous motif in non-oxygenic bacteria. The experimental means and resolved mechanism are relevant for numerous transmembrane protein-gated reactions.


Assuntos
Complexos de Proteínas Captadores de Luz/química , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/química , Synechocystis/química , Respiração Celular/genética , Transporte de Elétrons/genética , Elétrons , Cinética , Luz , Complexos de Proteínas Captadores de Luz/genética , Complexo de Proteína do Fotossistema II/genética , Synechocystis/genética
4.
Methods Mol Biol ; 1529: 3-19, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27914044

RESUMO

Computational protein design (CPD) has established itself as a leading field in basic and applied science with a strong coupling between the two. Proteins are computationally designed from the level of amino acids to the level of a functional protein complex. Design targets range from increased thermo- (or other) stability to specific requested reactions such as protein-protein binding, enzymatic reactions, or nanotechnology applications. The design scheme may encompass small regions of the proteins or the entire protein. In either case, the design may aim at the side-chains or at the full backbone conformation. Herein, the main framework for the process is outlined highlighting key elements in the CPD iterative cycle. These include the very definition of CPD, the diverse goals of CPD, components of the CPD protocol, methods for searching sequence and structure space, scoring functions, and augmenting the CPD with other optimization tools. Taken together, this chapter aims to introduce the framework of CPD.


Assuntos
Biologia Computacional/métodos , Engenharia de Proteínas/métodos , Proteínas , Simulação por Computador , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Relação Estrutura-Atividade
5.
Methods Mol Biol ; 1529: 21-94, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27914045

RESUMO

Computational protein design (CPD), a yet evolving field, includes computer-aided engineering for partial or full de novo designs of proteins of interest. Designs are defined by a requested structure, function, or working environment. This chapter describes the birth and maturation of the field by presenting 101 CPD examples in a chronological order emphasizing achievements and pending challenges. Integrating these aspects presents the plethora of CPD approaches with the hope of providing a "CPD 101". These reflect on the broader structural bioinformatics and computational biophysics field and include: (1) integration of knowledge-based and energy-based methods, (2) hierarchical designated approach towards local, regional, and global motifs and the integration of high- and low-resolution design schemes that fit each such region, (3) systematic differential approaches towards different protein regions, (4) identification of key hot-spot residues and the relative effect of remote regions, (5) assessment of shape-complementarity, electrostatics and solvation effects, (6) integration of thermal plasticity and functional dynamics, (7) negative design, (8) systematic integration of experimental approaches, (9) objective cross-assessment of methods, and (10) successful ranking of potential designs. Future challenges also include dissemination of CPD software to the general use of life-sciences researchers and the emphasis of success within an in vivo milieu. CPD increases our understanding of protein structure and function and the relationships between the two along with the application of such know-how for the benefit of mankind. Applied aspects range from biological drugs, via healthier and tastier food products to nanotechnology and environmentally friendly enzymes replacing toxic chemicals utilized in the industry.


Assuntos
Biologia Computacional , Engenharia de Proteínas , Proteínas , Biologia Computacional/história , Biologia Computacional/métodos , Simulação por Computador , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , História do Século XX , História do Século XXI , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Engenharia de Proteínas/história , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Software
6.
Structure ; 23(3): 527-541, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25703378

RESUMO

α Helices are a basic unit of protein secondary structure and therefore the interaction between helices is crucial to understanding tertiary and higher-order folds. Comparing subtle variations in the structural and sequence motifs between membrane and soluble proteins sheds light on the different constraints faced by each environment and elucidates the complex puzzle of membrane protein folding. Here, we demonstrate that membrane and water-soluble helix pairs share a small number of similar folds with various interhelical distances. The composition of the residues that pack at the interface between corresponding motifs shows that hydrophobic residues tend to be more enriched in the water-soluble class of structures and small residues in the transmembrane class. The latter group facilitates packing via sidechain- and backbone-mediated hydrogen bonds within the low-dielectric membrane milieu. The helix-helix interactome space, with its associated sequence preferences and accompanying hydrogen-bonding patterns, should be useful for engineering, prediction, and design of protein structure.


Assuntos
Proteínas de Membrana/química , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Solubilidade , Homologia Estrutural de Proteína
7.
Bioinformatics ; 31(1): 146-50, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25488929

RESUMO

MOTIVATION: The field of structural bioinformatics and computational biophysics has undergone a revolution in the last 10 years. Developments that are captured annually through the 3DSIG meeting, upon which this article reflects. RESULTS: An increase in the accessible data, computational resources and methodology has resulted in an increase in the size and resolution of studied systems and the complexity of the questions amenable to research. Concomitantly, the parameterization and efficiency of the methods have markedly improved along with their cross-validation with other computational and experimental results. CONCLUSION: The field exhibits an ever-increasing integration with biochemistry, biophysics and other disciplines. In this article, we discuss recent achievements along with current challenges within the field.


Assuntos
Pesquisa Biomédica/tendências , Biofísica/tendências , Biologia Computacional/tendências , Logro , Humanos
8.
Structure ; 20(5): 924-35, 2012 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-22579257

RESUMO

The complex hydrophobic and hydrophilic milieus of membrane-associated proteins pose experimental and theoretical challenges to their understanding. Here, we produce a nonredundant database to compute knowledge-based asymmetric cross-membrane potentials from the per-residue distributions of C(ß), C(γ) and functional group atoms. We predict transmembrane and peripherally associated regions from genomic sequence and position peptides and protein structures relative to the bilayer (available at http://www.degradolab.org/ez). The pseudo-energy topological landscapes underscore positional stability and functional mechanisms demonstrated here for antimicrobial peptides, transmembrane proteins, and viral fusion proteins. Moreover, experimental effects of point mutations on the relative ratio changes of dual-topology proteins are quantitatively reproduced. The functional group potential and the membrane-exposed residues display the largest energetic changes enabling to detect native-like structures from decoys. Hence, focusing on the uniqueness of membrane-associated proteins and peptides, we quantitatively parameterize their cross-membrane propensity, thus facilitating structural refinement, characterization, prediction, and design.


Assuntos
Proteínas de Membrana/química , Proteínas/química , Algoritmos , Bases de Dados Factuais , Interações Hidrofóbicas e Hidrofílicas , Bases de Conhecimento , Modelos Moleculares , Termodinâmica
10.
Protein Sci ; 20(7): 1256-64, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21563225

RESUMO

A hallmark of membrane protein structure is the large number of distorted transmembrane helices. Because of the prevalence of bends, it is important to not only understand how they are generated but also to learn how to predict their occurrence. Here, we find that there are local sequence preferences in kinked helices, most notably a higher abundance of proline, which can be exploited to identify bends from local sequence information. A neural network predictor identifies over two-thirds of all bends (sensitivity 0.70) with high reliability (specificity 0.89). It is likely that more structural data will allow for better helix distortion predictors with increased coverage in the future. The kink predictor, TMKink, is available at http://tmkinkpredictor.mbi.ucla.edu/.


Assuntos
Proteínas de Membrana/química , Prolina/química , Algoritmos , Bases de Dados de Proteínas , Modelos Moleculares , Estrutura Secundária de Proteína
11.
Annu Rev Phys Chem ; 62: 129-49, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21128762

RESUMO

From exponentially large numbers of possible sequences, protein design seeks to identify the properties of those that fold to predetermined structures and have targeted structural and functional properties. The interactions that confer structure and function involve intermolecular forces and large numbers of interacting amino acids. As a result, the identification of sequences can be subtle and complex. Sophisticated methods for characterizing sequences consistent with a particular structure have been developed, assisting the design of novel proteins. Developments in such computational protein design are discussed, along with recent accomplishments, ranging from the redesign of existing proteins to the design of new functionalities and nonbiological applications.


Assuntos
Simulação por Computador , Projeto Auxiliado por Computador/instrumentação , Proteínas de Membrana/química , Modelos Moleculares , Engenharia de Proteínas/instrumentação , Biologia Computacional/métodos , Modelos Estatísticos , Método de Monte Carlo , Dobramento de Proteína , Estrutura Secundária de Proteína/fisiologia , Relação Quantitativa Estrutura-Atividade
12.
Proc Natl Acad Sci U S A ; 107(34): 15075-80, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20689043

RESUMO

The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2(+) and 3(+) with a pK(a) near 6. A 1.65 A resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.


Assuntos
Vírus da Influenza A/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Cristalografia por Raios X , Feminino , Histidina/química , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Vírus da Influenza A/genética , Canais Iônicos/genética , Transporte de Íons , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oócitos/metabolismo , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas da Matriz Viral/genética , Xenopus
13.
Nature ; 442(7104): 827-30, 2006 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-16862124

RESUMO

Adjustment of catalytic activity in response to diverse ambient temperatures is fundamental to life on Earth. A crucial example of this is photosynthesis, where solar energy is converted into electrochemical potential that drives oxygen and biomass generation at temperatures ranging from those of frigid Antarctica to those of scalding hot springs. The energy conversion proceeds by concerted mobilization of electrons and protons on photoexcitation of reaction centre protein complexes. Following physicochemical paradigms, the rates of imperative steps in this process were predicted to increase exponentially with rising temperatures, resulting in different yields of solar energy conversion at the distinct growth temperatures of photosynthetic mesophiles and extremophiles. In contrast, here we show a meticulous adjustment of energy conversion rate, resulting in similar yields from mesophiles and thermophiles. The key molecular players in the temperature adjustment process consist of a cluster of hitherto unrecognized protein cavities and an adjacent packing motif that jointly impart local flexibility crucial to the reaction centre proteins. Mutations within the packing motif of mesophiles that increase the bulkiness of the amino-acid side chains, and thus reduce the size of the cavities, promote thermophilic behaviour. This novel biomechanical mechanism accounts for the slowing of the catalytic reaction above physiological temperatures in contradiction to the classical Arrhenius paradigm. The mechanism provides new guidelines for manipulating the acclimatization of enzymes to the ambient temperatures of diverse habitats. More generally, it reveals novel protein elements that are of potential significance for modulating structure-activity relationships in membrane and globular proteins alike.


Assuntos
Aclimatação , Fotossíntese , Proteínas/química , Proteínas/metabolismo , Temperatura Ambiente , Clorofila/metabolismo , Sequência Conservada , Cianobactérias/química , Cianobactérias/genética , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Elétrons , Transferência de Energia , Modelos Moleculares , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Maleabilidade , Conformação Proteica , Proteobactérias/química , Proteobactérias/metabolismo , Proteobactérias/efeitos da radiação , Relação Estrutura-Atividade
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