Controlling the Self-Assembly of Synthetic Metal-Coordinating Coiled-Coil Peptides

A distinct gap between the fields of peptide-based supramolecular assemblies and metal-organic frameworks (MOFs) exists. Despite recent efforts to explore this area of materials, many metal-directed peptide-based assemblies remain unpredictable, necessitating deeper study to establish robust design principles with which functional materials may be built. The profound unpredictability is not surprising, since peptide-based building blocks are more complex than others which have achieved success in organic and inorganic materials chemistry. The present study attempts to expand and solidify basic design rules for self-assembling metal-directed peptide-based architectures by first exerting control over the assembly of a serendipitously discovered supramolecular lattice. Recent published work has shown coiled-coil peptides containing one or more unnatural solvent-exposed terpyridine (Tpy) side chains can self-assemble to form supramolecular networks in the presence of copper (II) ion. Unexpected was the involvement of glutamic acid (Glu) side chains in the coordination motif (Tpy-Cu2+-Glu). Here, we seek to exert better control over these materials through systematic placement of both Tpy and Glu residues. Results are presented for six designed sequences, including one that manifests a hexagonal net rare in the materials literature. Collectively, these results demonstrate the power of harnessing peptide folding and metal coordination as driving forces for the formation of predictable supramolecular architectures

Driving conformational switching in de novo designed α-helical coiled-coils with novel molecular components

This research project is multidisciplinary in nature. It involves the use of biomolecular design –peptide design – and the synthesis of small organic compounds to generate conformational switching in peptide structures. In this thesis, we demonstrate that we can design and synthesize de novo peptide sequences with the necessary information to assemble as α-helical coiled-coil structures when associated with their corresponding peptide partners. In addition, some of the peptide structures are designed to form both α-helical coiled-coils and fibrous systems. Since we aim to promote conformational changes in the initial folding states of our peptide assemblies, the design of these individual sequences that we refer to as chassis peptides includes tuneable positions which after being modified will help these changes to happen. We make use of the Negishi reaction to synthesize unnatural amino acids – pyridyl-alanine analogues – for metal-binding investigations. The insertion of these novel amino acids into our self-assembling peptide systems provides different conformational changes depending on the positions in which these amino acids are inserted. These experiments are an attempt to form novel metal-based chiral biocatalysts. They also allow us to investigate to what extent peptide self-assembly can control metal binding and to what extent the metal binding can control peptide self-assembly. This research project also includes the synthesis of an azobenzene derivative for trans-to-cis and cis-to-trans photoisomerization. We successfully attached an azobenzene linker to three different coiled-coil forming peptide structures, exhibiting different switching efficiency in each. By using these photoswitches we induce conformational changes in the secondary structure of the peptide structures by the use of light. Some of these are reversible structural changes which makes it a potential power source for protein motors

Computational design and experimental characterization of metallopeptides as proteases for bioengineering applications

Enzymes are highly versatile catalysts present in biological systems and with high technological potential. Zinc metalloproteases are a major class of enzymes currently being employed in e.g. food, detergent, biopharmaceutical industries. In order to increase their robustness and range of biological and technological applications, metalloenzymes can be redesigned by exploring the chemical versatility of different metals along with protein sequence modifications. In this work, the computational design of new zinc metalloproteases was approached to test if proteolytic activity can be recapitulated in small scaffolds tailored for bioengineering applications. Structural and dynamical aspects of metalloproteases were first addressed to identify catalytically-relevant interactions. Models of the active site were developed to screen a set of 43 small scaffolds with the Rosetta software for their ability to recapitulate the native enzyme functionality. Two candidate scaffolds were selected for enzyme design and experimental characterization, namely the Sp1 zinc finger 2 and the villin headpiece subdomain. While metal coordination was achieved (binding constants KZnP,app in the 105 M-1 range), the scaffolds presented low stabilities (thermal unfolding bellow 50 °C) most likely due to perturbations introduced by the 4 to 10 sequence modifications. The metallopeptides presented catalytic activity towards ester substrates within the range of values found for other small scaffolds in the literature (second-order rate constants k2 in the 10-1 M-1s-1 range). The design approach developed in this work was successful in achieving catalytically-active metallopeptides, although target metalloprotease activity could not be achieved. Molecular dynamics simulations in microsecond regimes were subsequently used to detect design flaws related with high scaffold flexibility. This work contributes to the improvement of the computational enzyme design approaches by pointing out the need for a dynamical treatment of the designs in longer time-scales, and through the development of fast methods to rank and evaluate a large number of potential biocatalysts.Enzimas são versáteis catalisadoras presentes em sistemas biológicos e com elevado potencial tecnológico. Metaloproteases de zinco são uma classe de enzimas com aplicação corrente na indústria e.g. alimentar, biofarmacêutica e detergentes. A versatilidade química de diferentes metais combinada com modificações na sequência de metaloenzimas podem ser exploradas de forma a aumentar a sua robustez e leque de aplicações biológicas e tecnológicas, Neste trabalho, novos metaloproteases de zinco foram desenhados computacionalmente de forma a testar se atividade proteolítica pode ser reproduzida em proteínas pequenas adaptadas para bioengenharia. Análise de aspetos estruturais/dinâmicos de metaloproteases permitiram identificar interações cataliticamente relevantes. Modelos do centro ativo de zinco foram desenvolvidos para examinar com o software Rosetta um conjunto de 43 pequenas estruturas quanto ao seu potencial em recapitular a função nativa de enzimas. Duas estruturas foram selecionadas para design e caracterização experimental, nomeadamente o “zinc-finger” 2 da proteína Sp1 e o subdomínio cabeça da vilina. Embora coordenação com o metal tenha sido alcançada (constantes de afinidade KZnP,app na ordem 105 M-1), as estruturas apresentam baixa estabilidade (temperatura de desnaturação inferior a 50 °C), refletindo perturbações provavelmente causadas por 4-10 modificações de sequência. Os metalopéptidos apresentam actividade catalítica para ésteres semelhante aos valores de literatura obtidos para outras pequenas estruturas (constantes de segunda ordem k2 na ordem 10-1 M-1s-1). A metodologia desenvolvida neste trabalho foi bem sucedida em desenhar metalopéptidos catalíticos, embora a atividade alvo de metaloprotease não tenha sido alcançada. Simulações de dinâmica molecular na escala de microsegundo foram usadas posteriormente para detectar falhas nos designs relacionadas com elevada flexibilidade estrutural. Este trabalho contribui para o melhoramento de métodos de design computacional de enzimas, ao demonstrar a necessidade de considerar aspectos dinâmicos dos designs em escalas de tempo maiores, e no desenvolvimento de métodos rápidos para classificar e avaliar um vasto leque de potenciais biocatalisadores

Design of protein-nanomaterial hybrids as tools for sensing, imaging and bioelectronics

217 p.El diseño de proteínas permite construir herramientas nanotecnológicas adaptadas para su uso en campos como la biomedicina o la industria. Las proteínas de repetición CTPR son una buena opción para desarrollar nano-herramientas dada su estructura modular y tolerancia a mutaciones, lo que permite combinar módulos funcionalizados sin comprometer la estabilidad de la proteína. Además, las proteínas CTPR pueden modificarse para desarrollar módulos que coordinan metales, lo que permite la unión de nanomateriales metálicos con propiedades interesantes como las nanopartículas de oro, o la síntesis de nanocristales metálicos in situ. En la presente tesis doctoral se propone un sistema modular de proteínas CTPR funcionalizadas con nanomateriales metálicos para su aplicación como herramientas nanotecnológicas en sensórica, imagen y bioelectrónica. Para ello, primero se establece un diseño de CTPR con residuos de coordinación de metales y se estudia en profundidad las propiedades fotoluminiscentes que emergen de nanocristales de oro coordinados a dichas CTPR. A continuación, se elaboran diseños de CTPR coordinando nanomateriales metálicos y se aplican como sensores de parámetros ambientales, como la temperatura o la presencia de iones metálicos; como sondas fluorescentes para detección correlativa de orgánulos celulares usando microscopía de fluorescencia y fluorescencia de rayos-X; y como bloques de construcción para elaborar biomateriales conductores

Synthesis and Applications of Dirhodium Metallopeptides

The work describes the development of a new class of synthetic metallopeptides that features a dirhodium metal center. Combination of peptide and dirhodium properties leads to unique effects on peptide structure, peptide-protein interactions, and metal catalytic activity aimed at small molecule as well as protein substrates. Dirhodium is directly bound to carboxylate side chains of aspartate or glutamate yielding kinetically inert coordination complexes. This improves stability, allows purification and provides enhanced biocompatibility. Bridging of two side chains in the same sequence enables control of the peptide secondary structure. Dirhodium metallopeptides are applied to regulate coiled coil dimerization, stabilize and induce helical secondary structure, catalyze enantioselective organometallic transformation, and serve as ligands for proteins. These results lead to the development of hybrid organic-inorganic therapeutic agents, biological probes for study of protein-protein interactions, and enantioselective metallopeptide catalysis

Solid-state NMR spectroscopy

M.H. acknowledges support by National Institutes of Health (NIH) grant GM066976.Solid-state nuclear magnetic resonance (NMR) spectroscopy is an atomic-level method used to determine the chemical structure, three-dimensional structure, and dynamics of solids and semi-solids. This Primer summarizes the basic principles of NMR as applied to the wide range of solid systems. The fundamental nuclear spin interactions and the effects of magnetic fields and radiofrequency pulses on nuclear spins are the same as in liquid-state NMR. However, because of the anisotropy of the interactions in the solid state, the majority of high-resolution solid-state NMR spectra is measured under magic-angle spinning (MAS), which has profound effects on the types of radiofrequency pulse sequences required to extract structural and dynamical information. We describe the most common MAS NMR experiments and data analysis approaches for investigating biological macromolecules, organic materials, and inorganic solids. Continuing development of sensitivity-enhancement approaches, including 1H-detected fast MAS experiments, dynamic nuclear polarization, and experiments tailored to ultrahigh magnetic fields, is described. We highlight recent applications of solid-state NMR to biological and materials chemistry. The Primer ends with a discussion of current limitations of NMR to study solids, and points to future avenues of development to further enhance the capabilities of this sophisticated spectroscopy for new applications.PostprintPeer reviewe

Biocatalysts based on peptide and peptide conjugate nanostructures

Peptides and their conjugates (to lipids, bulky N-terminals, or other groups) can self-assemble into nanostructures such as fibrils, nanotubes, coiled coil bundles, and micelles, and these can be used as platforms to present functional residues in order to catalyze a diversity of reactions. Peptide structures can be used to template catalytic sites inspired by those present in natural enzymes as well as simpler constructs using individual catalytic amino acids, especially proline and histidine. The literature on the use of peptide (and peptide conjugate) α-helical and β-sheet structures as well as turn or disordered peptides in the biocatalysis of a range of organic reactions including hydrolysis and a variety of coupling reactions (e.g., aldol reactions) is reviewed. The simpler design rules for peptide structures compared to those of folded proteins permit ready ab initio design (minimalist approach) of effective catalytic structures that mimic the binding pockets of natural enzymes or which simply present catalytic motifs at high density on nanostructure scaffolds. Research on these topics is summarized, along with a discussion of metal nanoparticle catalysts templated by peptide nanostructures, especially fibrils. Research showing the high activities of different classes of peptides in catalyzing many reactions is highlighted. Advances in peptide design and synthesis methods mean they hold great potential for future developments of effective bioinspired and biocompatible catalysts

Structural analysis of cross α-helical nanotubes provides insight into the designability of filamentous peptide nanomaterials

The exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili