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1.
J Am Chem Soc ; 142(1): 233-241, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31815456

RESUMO

Self-assembled peptide micelles and fibers demonstrate unique control over the photophysical properties of the bound, light-activated chromophore, zinc protoporphyrin IX, (PPIX)Zn. Micelles encapsulate either a mixture of uncoordinated and coordinated (PPIX)Zn or all coordinated depending on the ratio of peptide/porphyrin. As the ratio increases toward a 1:1 micelle/porphyrin ratio, providing the chromophore with a discrete coordination environment reminiscent of unstructured proteins, the micelles favor triplet formation. Fibers, however, promote a linear array of porphyrin molecules that dictates exciton hopping and excimer formation at ratios as high as 60:1, peptide/porphyrin. However, even in fibers, the formation of the triplet species increases with increasing peptide/porphyrin ratio due to increased spatial separation between neighboring chromophores facilitating intersystem crossing. Full characterization of the micelles structures and comparison to the fibers lead to the comparison with natural systems and the ability to control the excited populations that have utility in photocatalytic processes. In addition, the incorporation of a second chromophore, heme, yields an electron transfer pathway in both micelles and fibers that highlights the utility of the peptide assemblies when engineering multichromophore arrays as inspired by natural, photosynthetic proteins.

2.
Nanoscale ; 11(12): 5412-5421, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30855041

RESUMO

To take peptide materials from predominantly structural to functional assemblies, variations in cofactor binding sites must be engineered and controlled. Here, we have employed the peptide sequence c16-AHX3K3-CO2H where X3 represents the aliphatic structural component of the peptide design that dictates ß-sheet formation and upon self-assembly yields a change in the overall microenvironment surrounding the Zn protoporphyrin IX ((PPIX)Zn) binding site. All peptides studied yield ß-sheet rich nanofibers highlighting the materials' resiliency to amino acid substitution. We highlight that the (PPIX)Zn binding constants correlate strongly with amino acid side chain volume, where X = L or I yields the lowest dissociation constant values (KD). The resulting microenvironment highlights the materials' ability to control interchromophore electronic interactions such that slip-stacked cofacial arrangements are observed via exciton splitting in UV/visible and circular dichroism spectroscopy. Steady state and time-resolved photoluminescence suggests that greater interchromophore packing yields larger excimer populations and corresponding longer excimer association lifetimes (τA) which directly translates to shorter exciton diffusion lengths. In comparison to synthetic porphyrin molecular assemblies, this work demonstrates the ability to employ the peptide assembly to modulate the degree of cofactor arrangement, extent of excimer formation, and the exciton hopping rates all while in a platform amenable for producing polymer-like materials.


Assuntos
Nanofibras/química , Peptídeos/química , Protoporfirinas/química , Sítios de Ligação , Dicroísmo Circular , Microscopia Eletrônica de Transmissão , Ligação Proteica , Conformação Proteica em Folha beta , Espectrofotometria
3.
Org Biomol Chem ; 15(32): 6725-6730, 2017 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-28782067

RESUMO

Nature guides the flow of electrons in biological systems with the assistance of multi-heme proteins called cytochromes. In an effort to understand natures approach to developing electronic systems, three peptides that are compositionally identical but sequentially distinct have been designed to study the impact of morphology and hydrophobicity on heme coordination and function.


Assuntos
Citocromos/síntese química , Elétrons , Heme/química , Sítios de Ligação , Citocromos/química , Interações Hidrofóbicas e Hidrofílicas , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química
4.
ACS Nano ; 11(9): 9112-9118, 2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28817256

RESUMO

Light-harvesting biomaterials are an attractive target in photovoltaics, photocatalysis, and artificial photosynthesis. Through peptide self-assembly, complex nanostructures can be engineered to study the role of chromophore organization during light absorption and energy transport. To this end, we demonstrate the one-dimensional transport of excitons along naturally occurring, light-harvesting, Zn-protoporphyrin IX chromophores within self-assembled peptide-amphiphile nanofibers. The internal structure of the nanofibers induces packing of the porphyrins into linear chains. We find that this peptide assembly can enable long-range exciton diffusion, yet it also induces the formation of excimers between adjacent molecules, which serve as exciton traps. Electronic coupling between neighboring porphyrin molecules is confirmed by various spectroscopic methods. The exciton diffusion process is then probed through transient photoluminescence and absorption measurements and fit to a model for one-dimensional hopping. Because excimer formation impedes exciton hopping, increasing the interchromophore spacing allows for improved diffusivity, which we control through porphyrin doping levels. We show that diffusion lengths of over 60 nm are possible at low porphyrin doping, representing an order of magnitude improvement over the highest doping fractions.


Assuntos
Nanofibras/química , Peptídeos/química , Protoporfirinas/química , Tensoativos/química , Luminescência , Modelos Moleculares , Nanofibras/ultraestrutura
5.
J Am Chem Soc ; 139(25): 8497-8507, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28505436

RESUMO

Self-assembling peptide materials have gained significant attention, due to well-demonstrated applications, but they are functionally underutilized. To advance their utility, we use noncovalent interactions to incorporate the biological cofactor heme-B for catalysis. Heme-proteins achieve differing functions through structural and coordinative variations. Here, we replicate this phenomenon by highlighting changes in heme reactivity as a function of coordination, sequence, and morphology (micelles versus fibers) in a series of simple peptide amphiphiles with the sequence c16-xyL3K3-CO2H where c16 is a palmitoyl moiety and xy represents the heme binding region: AA, AH, HH, and MH. The morphology of this peptide series is characterized using transmission electron and atomic force microscopies as well as dynamic light scattering. Within this small library of peptide constructs, we show that three spectroscopically (UV/visible and electron paramagnetic resonance) distinct heme environments were generated: noncoordinated/embedded high-spin, five-coordinate high-spin, and six-coordinate low-spin. The resulting material's functional dependence on sequence and supramolecular morphology is highlighted 2-fold. First, the heme active site binds carbon monoxide in both micelles and fibers, demonstrating that the heme active site in both morphologies is accessible to small molecules for catalysis. Second, peroxidase activity was observed in heme-containing micelles yet was significantly reduced in heme-containing fibers. We briefly discuss the implications these findings have in the production of functional, self-assembling peptide materials.


Assuntos
Heme/química , Peptídeos/química , Catálise , Domínio Catalítico , Micelas , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Tensoativos/química
6.
Chem Sci ; 8(1): 316-324, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28261441

RESUMO

Natural selection in photosynthesis has engineered tetrapyrrole based, nanometer scale, light harvesting and energy capture in light-induced charge separation. By designing and creating nanometer scale artificial light harvesting and charge separating proteins, we have the opportunity to reengineer and overcome the limitations of natural selection to extend energy capture to new wavelengths and to tailor efficient systems that better meet human as opposed to cellular energetic needs. While tetrapyrrole cofactor incorporation in natural proteins is complex and often assisted by accessory proteins for cofactor transport and insertion, artificial protein functionalization relies on a practical understanding of the basic physical chemistry of protein and cofactors that drive nanometer scale self-assembly. Patterning and balancing of hydrophobic and hydrophilic tetrapyrrole substituents is critical to avoid natural or synthetic porphyrin and chlorin aggregation in aqueous media and speed cofactor partitioning into the non-polar core of a man-made water soluble protein designed according to elementary first principles of protein folding. This partitioning is followed by site-specific anchoring of tetrapyrroles to histidine ligands strategically placed for design control of rates and efficiencies of light energy and electron transfer while orienting at least one polar group towards the aqueous phase.

7.
Nat Commun ; 7: 12367, 2016 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-27554944

RESUMO

Understanding the role of water in governing the kinetics of the self-assembly processes of amphiphilic peptides remains elusive. Here, we use a multistage atomistic-coarse-grained approach, complemented by circular dichroism/infrared spectroscopy and dynamic light scattering experiments to highlight the dual nature of water in driving the self-assembly of peptide amphiphiles (PAs). We show computationally that water cage formation and breakage near the hydrophobic groups control the fusion dynamics and aggregation of PAs in the micellar stage. Simulations also suggest that enhanced structural ordering of vicinal water near the hydrophilic amino acids shifts the equilibrium towards the fibre phase and stimulates structure and order during the PA assembly into nanofibres. Experiments validate our simulation findings; the measured infrared O-H bond stretching frequency is reminiscent of an ice-like bond which suggests that the solvated water becomes increasingly ordered with time in the assembled peptide network, thus shedding light on the role of water in a self-assembly process.


Assuntos
Peptídeos/química , Tensoativos/química , Água/química , Dicroísmo Circular , Difusão Dinâmica da Luz , Interações Hidrofóbicas e Hidrofílicas , Micelas , Modelos Moleculares , Simulação de Dinâmica Molecular , Nanofibras/química , Agregados Proteicos , Multimerização Proteica , Espectrofotometria Infravermelho
8.
Biochim Biophys Acta ; 1857(5): 513-521, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26423266

RESUMO

Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7×10(6) M(-1) s(-1) to 1.2×10(9) M(-1) s(-1) follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and -19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.


Assuntos
Citocromos c/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Citocromos c/genética , Citocromos c/metabolismo , Difusão , Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Fotólise , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
9.
J Am Chem Soc ; 136(8): 3192-9, 2014 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-24495285

RESUMO

Timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes, including the oxidoreductases and allied transport and signaling proteins. Likewise, ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man-made, first-principle protein constructs intended to operate in vitro or in vivo. As one of the most common catalytic cofactors in biology, we have chosen heme B, along with its chemical analogues, to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4-α-helix proteins. We compare five elementary synthetic designs (maquettes) and the natural cytochrome b562 that differ in oligomeric forms, apo- and holo-tertiary structural stability; qualities that we show can either assist or hinder assembly. The cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic. With progressive removal of identified barriers, we achieve maquette assembly rates as fast as native cytochrome b562, paving the way to in vivo assembly of man-made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways.


Assuntos
Heme/química , Proteínas/síntese química , Cinética , Estrutura Secundária de Proteína , Proteínas/química , Espectrofotometria Ultravioleta , Termodinâmica
10.
Nat Chem Biol ; 9(12): 826-833, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24121554

RESUMO

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.


Assuntos
Oxirredutases/metabolismo , Proteínas/química , Heme/química , Heme/metabolismo , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredutases/química , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos
11.
Biochem Soc Trans ; 40(3): 561-6, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22616867

RESUMO

The study of natural enzymes is complicated by the fact that only the most recent evolutionary progression can be observed. In particular, natural oxidoreductases stand out as profoundly complex proteins in which the molecular roots of function, structure and biological integration are collectively intertwined and individually obscured. In the present paper, we describe our experimental approach that removes many of these often bewildering complexities to identify in simple terms the necessary and sufficient requirements for oxidoreductase function. Ours is a synthetic biology approach that focuses on from-scratch construction of protein maquettes designed principally to promote or suppress biologically relevant oxidations and reductions. The approach avoids mimicry and divorces the commonly made and almost certainly false ascription of atomistically detailed functionally unique roles to a particular protein primary sequence, to gain a new freedom to explore protein-based enzyme function. Maquette design and construction methods make use of iterative steps, retraceable when necessary, to successfully develop a protein family of sturdy and versatile single-chain three- and four-α-helical structural platforms readily expressible in bacteria. Internally, they prove malleable enough to incorporate in prescribed positions most natural redox cofactors and many more simplified synthetic analogues. External polarity, charge-patterning and chemical linkers direct maquettes to functional assembly in membranes, on nanostructured titania, and to organize on selected planar surfaces and materials. These protein maquettes engage in light harvesting and energy transfer, in photochemical charge separation and electron transfer, in stable dioxygen binding and in simple oxidative chemistry that is the basis of multi-electron oxidative and reductive catalysis.


Assuntos
Oxirredutases/síntese química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/síntese química , Biologia Sintética/métodos , Oxirredução , Oxirredutases/química , Proteínas Recombinantes/química
12.
Nature ; 458(7236): 305-9, 2009 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-19295603

RESUMO

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.


Assuntos
Proteínas de Transporte/síntese química , Proteínas de Transporte/metabolismo , Oxigênio/metabolismo , Engenharia de Proteínas , Transporte Biológico , Monóxido de Carbono/metabolismo , Proteínas de Transporte/química , Desenho de Fármacos , Globinas/química , Ácido Glutâmico/metabolismo , Heme/metabolismo , Histidina/metabolismo , Humanos , Cinética , Ligantes , Proteínas do Tecido Nervoso/química , Neuroglobina , Oxirredução , Estrutura Secundária de Proteína , Rotação , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por Substrato , Água/análise , Água/metabolismo
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