Ratcheting synthesis

Synthetic chemistry has traditionally relied on reactions between reactants of high chemical potential and transformations that proceed energetically downhill to either a global or local minimum (thermodynamic or kinetic control). Catalysts can be used to manipulate kinetic control, lowering activation energies to influence reaction outcomes. However, such chemistry is still constrained by the shape of one-dimensional reaction coordinates. Coupling synthesis to an orthogonal energy input can allow ratcheting of chemical reaction outcomes, reminiscent of the ways that molecular machines ratchet random thermal motion to bias conformational dynamics. This fundamentally distinct approach to synthesis allows multi-dimensional potential energy surfaces to be navigated, enabling reaction outcomes that cannot be achieved under conventional kinetic or thermodynamic control. In this Review, we discuss how ratcheted synthesis is ubiquitous throughout biology and consider how chemists might harness ratchet mechanisms to accelerate catalysis, drive chemical reactions uphill and programme complex reaction sequences.<br/

ICR ANNUAL REPORT 2022 (Volume 29)[All Pages]

This Annual Report covers from 1 January to 31 December 202

Adapting Proteins for Clinical and Industrial Use

Enzymes are powerful tools that are capable of catalyzing reactions with high specificity and efficiency. Many naturally occurring enzymes are used as tools in industry and medicine, from food additives to pharmaceuticals to biofuel production. Protein engineering is used to make enzymes more readily available, to gain new or improved catalytic function, or optimize properties such as thermostability and enantioselectivity. In this thesis we will discuss the variety of ways proteins can be modified to one day be able to be put to use in industrial and clinical settings. My goal has been to optimize proteins for industrial and clinical use by either a) understanding how the protein evolved so that the knowledge can be applied to other protein designs, b) finding new ways to express proteins so that they can be isolated and used more easily in industrial and clinical settings, and c) creating new proteins that can be used for industrial and clinical settings. In Chapter 2, through protein engineering, a new protein was made that selectively binds lanthanides over its natural metal, calcium, with high affinity. This provides us with important information into how metalloproteins evolve metal specificity, which can be applied to designing new metalloproteins. In Chapter 3, we investigate a possible application of this protein in therapeutics by attaching an antibody to the protein structure that is specific for Hepatocellular Carcinoma cells and using the protein to bind and deliver radioisotopes. This would allow us to not only create a new treatment option for this variety of cancer, but also allow us to optimize the treatment more effectively and easily through protein evolution. In Chapter 4, we discuss how changing the expression vector of an industrially relevant enzyme, formate dehydrogenase, allows it to be synthesized and secreted by Pichia pastoris and can be an important first step in creating a self-sustaining and environmentally safe method of producing methanol biofuel. Chapter 5 focuses on investigating a new method of evolving designed proteins that uses NMR by analyzing known mutations in an extensively evolved protein. This would one day allow potential enzymes to be optimized readily and cheaply for commercial use. Finally, Chapter 6 will focus on using amino acids as an alternative to traditional chiral ligands in organometallic synthesis. These new ligands would be much cheaper to make, easier to produce, and have the added benefit of forming fibrils that would make them easy to collect and attach to surfaces. These studies contribute to a better understanding of the variety of methods proteins can be optimized for use in our daily lives

Gold Binding Peptide Fused Alkaline Phosphatase For Real Rime Monitoring Of Calcium Phosphate Biomineralization

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2012Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2012Anorganik yüzeylere özgül olarak bağlanan genetik yöntemlerle tasarlanmış peptitler, doğadan esinlenen çok işlevli sistemlerin oluşturulması için bir önemli üstünlükler sağlamaktadır. Kısa amino asit zincirlerinden oluşan bu peptitler moleküler bağlayıcılar olarak geleneksel yöntemlerin karşılaştığı güçlüklere çözüm olarak kullanılabilecek işlevsel biyomoleküllerdir. Bu çalışmada beş sıralı tekrar biçiminde altına özgü bağlanan peptitin alkalin fosfataz enzimine moleküler biyoloji yöntemleriyle eklenmiş yapısı, kalsiyum fosfat mineralizasyonunun gerçek zamanlı takip edilmesi amacıyla kullanılmıştır. Kalsiyum fosfat kemik ve diş gibi sert dokuların ana maddesi olan önemli bir biyomineral olarak hem omurgalı hem de omurgasız canlılarda farklı kristal yapılarda bulunmaktadır. Kuramsal açıdan olduğu kadar tedavi alanında pratik bakımından da önemli olmasından dolayı kalsiyum fosfatın hem oluşum hem de kristal faz geçişleri hakkında birçok çalışma yürütülmektedir. Alkalin fosfataz enzimi fosfat grubu içeren kimyasal yapılarndan hidroliz tepkimesi ile anorganik fosfat grubu açığa çıkmasını sağlama özelliğine sahiptir. Bu nedenle mineral oluşumu çalışmalarında biyominerallerin üretilmesi için söz konusu enzimin bu tür tepkimelerinden esinlenilmektedir. Yüzey plazmonik rezonans spektroskopisi moleküler etkileşimlerin gerçek zamanlı incelenmesini sağlayan çok hassas bir karakterizasyon yöntemidir. Ayrıca SPR proteinlerin katı yüzeylerle özellikle altın yüzeyle etkileşimini incelemek açısında yararlı bir tekniktir. Bu çalışmada GEPI molekülleriyle güçlendirilmiş altına özgü bağlanma özelliği olan alkalin fosfataz enzimi SPR yöntemi kullanılarak mineral oluşumunun gerçek zamanlı incelenmesi amaçlanmıştır. Bu deneysel çalışmalarda izlenen yöntemler hem biyomineral oluşumunun gerçek zamanlı takibine olanak sağlamış hem de genetik yöntemlerle tasarlanan çok işlevli alkalin fosfataz enziminin altına özgü bağlanma özellikleri incelenme imkânı sunmuştur. Ayrıca doğal enzime kıyasla GEPI molekülleriyle işlevselleştirilmiş alkalin fosfatazın daha yüksek aktivite gösterdiği bulunmuştur. Elde edilen sonuçlara göre GEPI bağlı alkalin fosfataz altın yüzey üzerinde biyomineral oluşumunu tetikleme özelliğine ek olarak kalsiyum fosfat oluşumunun yüzey plazmonik rezonans yöntemi ile takibine olanak sağlamıştır. Yüzey plazmonik resonans spektroskopisinin biyomineral oluşumu hakkında uzun süredir tartışmalı bir konu olan kalsiyum fosfatın olası kristal faz geçişlerini destekleyen veriler sağladığı düşünülmektedir.Genetically engineered peptides for inorganics (GEPIs) that have the ability to bind specifically to an inorganic surface provide important advantageous for constructing multifunctional systems which inspired from natue. These short amino acid sequences serve as linker molecules in order to realize striking nanobiotechnological applications. GEPIs fused proteins serve as multifunctional biomolecules which overcome limitations of traditional immobilization techniques. In this dissertation, alkaline phosphatase enzyme fused to a genetically enginneered gold binding peptide with five tandem repeat (5GBP1-AP) was used for real time monitoring of calcium phosphate biomineralization. Calcium phosphate as being the main component of hard tissues such as bone and teeth is a crucial biomineral which is found in different crystal structures in both vertebrates and invertebrates. Calcium phosphate is widely studied in order to shed light of its formation and crystal phase transitions that are important for theoretically and practically for medical applications. Alkaline phosphatase has the ability to catalyze the hydrolizing reaction of phosphate containing molecules result to release inorganic phospahate group to the environment. For this reason, it is used in mineralization studies to mimic this function for biomineral formation. Surface plasmon resonance (SPR) spectroscopy is a very sensitive characterization method to analyze molecular interactions in real-time. In addition SPR serve as valuable technique to study protein interactions with solid surface that is commonly gold substrate. GEPI fused alkaline phosphate which have high affinity to gold surafec was analyzed by SPR in order to progress biomineral formation in real-time. The advantage of this method is the ability to provide both monitoring of calcium phosphate formation in real-time and analyzing gold binding property of bifunctional enzyme. In addition GEPI fused AP was found to have higher enzymatic activity according to wild type enzyme. Results indicated that 5GBP1-AP induced biomineral formation upon adsorption onto gold substate and allow to progress biomineral formation by surface plasmon resonance spectroscopy. SPR results are thought to be promising with the possible indications about the crystal phase transition of calcium phosphate which is a long debated issue about biomineralization.Yüksek LisansM.Sc

Magnetism, FeS colloids, and Origins of Life

A number of features of living systems: reversible interactions and weak bonds underlying motor-dynamics; gel-sol transitions; cellular connected fractal organization; asymmetry in interactions and organization; quantum coherent phenomena; to name some, can have a natural accounting via $physical$ interactions, which we therefore seek to incorporate by expanding the horizons of `chemistry-only' approaches to the origins of life. It is suggested that the magnetic 'face' of the minerals from the inorganic world, recognized to have played a pivotal role in initiating Life, may throw light on some of these issues. A magnetic environment in the form of rocks in the Hadean Ocean could have enabled the accretion and therefore an ordered confinement of super-paramagnetic colloids within a structured phase. A moderate H-field can help magnetic nano-particles to not only overcome thermal fluctuations but also harness them. Such controlled dynamics brings in the possibility of accessing quantum effects, which together with frustrations in magnetic ordering and hysteresis (a natural mechanism for a primitive memory) could throw light on the birth of biological information which, as Abel argues, requires a combination of order and complexity. This scenario gains strength from observations of scale-free framboidal forms of the greigite mineral, with a magnetic basis of assembly. And greigite's metabolic potential plays a key role in the mound scenario of Russell and coworkers-an expansion of which is suggested for including magnetism.Comment: 42 pages, 5 figures, to be published in A.R. Memorial volume, Ed Krishnaswami Alladi, Springer 201

Clonable selenium nanoparticle in action: high resolution localization of FtsZ using electron tomography, A

2021 Spring.Includes bibliographical references.A meaningful understanding of biochemistry requires that we understand the function of proteins, which is heavily dependent on their structure and location within an organism. As the Resolution Revolution of cryo-electron microscopy gains unprecedented ground largely due to the recent development of commercially available direct electron detectors, energy filters, and high-end computation, thousands of protein structures have been solved at atomic or near-atomic resolution, with the highest resolution structure to date being solved at 1.2 Å. A major challenge that has limited the broad use of cryo-electron tomography (cryo-ET) is locating a protein of interest in an organism, as no commercially available high-contrast markers which can be generated in vivo exist. Herein, we present a breakthrough study which aims to solve this problem by synthesizing high contrast metal nanoparticles labeling desired proteins in situ. We isolated a Glutathione Reductase-like Metalloid Reductase (GRLMR), which can reduce selenite and selenate into selenium nanoparticles (SeNPs), from Pseudomonas moraviensis stanleyae found in the roots of a Se hyperaccumulator Stanleya pinnata, or Desert Princes' Plume. A recombinant variant, denoted as a clonable Selenium NanoParticle (cSeNP), was fused to filamentous temperature sensitive protein Z (FtsZ), and the chimera was expressed in vivo using a T7 expression system in model organism E. coli for a proof-of-concept study. Because the SeNPs biogenically produced are amorphous, they exist in a quasistable state and are composed of polymeric Sen in the form of chains and rings that are constantly breaking and reforming. To stabilize the particles during cellular preservation ex aqua, a disproportionation-like reaction can be done either in vivo or as a post-fixation step to form crystalline metal selenide (MSe) NPs that can withstand the processing liquids used. Thereafter, electron tomography was used to acquire a tilt series that was reconstructed into a tomogram and segmented using IMOD, generating a model representing MSeNPs labeling FtsZ filaments. As such, we have demonstrated the potential of using cSeNP as a high resolution marker for cryo-ET. While our study relied on traditional preservation and embedment techniques, we anticipate that for cells preserved via vitrification, cloned SeNPs can be used without subsequent transformation to MSeNPs, as the amorphous particles are stable in aqueous media. Prospectively, we expect that clonable nanoparticle technology will revolutionize cryo-ET, allowing us to localize proteins in vivo at high resolution while maintaining organism viability through metal immobilization. Furthermore, this technique can be expanded to other imaging modalities, such as light microscopy and X-ray tomography, through the discovery and engineering of other clonable nanoparticles

Understanding the Structure-Function Relationship in Peptide-Enabled High Entropy Alloy Nanocatalysts

The structural complexity in high entropy alloy nanocatalysts (HEAs), afforded by the homogeneous mixing of five or more elements, has resulted in a limited understanding about the origin of their promising electrocatalytic properties. This thesis investigates the structure-function relationship in HEAs using advanced material characterization techniques. At first, a methodology for resolving the atomic-scale structure of peptide-enabled HEAs was developed using high-energy X-ray diffraction (HE-XRD) coupled with atomic pair distribution function (PDF) and reverse Monte Carlo (RMC) simulations, yielding structure models over the length scale of HEAs. Coordination analysis of the structure models revealed a multifunctional interplay of geometric and electronic attributes of surface atoms in HEAs that was responsible for the catalytic activity enhancement during the methanol electrooxidation reaction. Using the methodology for resolving the atomic scale structure of HEAs and peptide sequence engineering, the structure-function relationship of model PtPdAuCoSn HEAs during ethanol electrooxidation reaction (EOR) was studied. Compositional analysis of the PtPdAuCoSn HEA structure models revealed distinct miscibility characteristics that were attributed to the unique biotic-abiotic interactions. Analysis of the structure models identified the rapid dehydrogenation of CH3CHO intermediate into CH3COads in an optimized adsorption configuration as the contributing factor for the high selectivity towards CH3COO- in PtPdAuCoSn HEAs. Armed with these insights, a study was designed for understanding the effect of changing the concentration of Pt in the structure-function relationship of PtPdAuCoSn HEAs using spatiotemporal structural insights from in-situ PDF. The structure models demonstrated a degree of metastability as a function of their corresponding configurational entropy. Analysis of the structure models revealed that high selectivity towards CH3COO- in PtPdAuCoSn HEAs during EOR originates from the enhanced distribution of Pd and Co surface atoms. In summary, this thesis uses atomic PDF and RMC simulations to draw structure-function correlations in HEAs, presenting a path forward for developing strategies for the rational design of HEAs. Through collaborative efforts from theoreticians and experimentalists, the methodology presented here can form the basis for accelerating the discovery of promising HEA configurations for emerging electrocatalytic applications

The Design of Heteromeric and Metal-binding Alpha-Helical Barrels

Introduction: The field of protein design has drastically evolved over the past four years. Both the protein folding problem, which involves predicting the 3D arrangement of atoms from a given sequence of amino acids, and its inverse, have been technically solved after 50 years. However, the black box nature of the tools developed to address these problems limits our comprehension of protein folding and dynamics. Harnessing this knowledge could revolutionise sectors such as drug design, disease diagnosis, energy transfer, and material science. This work focuses on the rational design of a protein scaffold called coiled coils, positioning them as a model for advancing our control and understanding of proteins.Results: In this thesis, we navigate the uncharted territory of coiled coils with reduced symmetry. We generate novel A3B3 hexameric α-helical barrels with both parallel and antiparallel helix orientations, expanding understanding of coiled-coil assemblies and introducing new scaffolds. Utilising these assemblies, we create covalently attached bipyridyl functional groups situated within the barrel cores, capable of chelating iron and ruthenium ions. Additionally, we develop intrinsically disordered peptide sequences that assemble only upon the introduction of specific metal ions. This can be applied for both metal sensing, as well as metal mediated sensing of other ligands.Conclusions: This research advances the field of protein design through the generation of novel α-helical barrels and the development of coiled-coil assemblies with innovative functionalities. Our work has allowed for new potential applications in bio-sensing and catalysis and has further demonstrated the broad versatility of coiled-coil scaffolds.Implications: This study illuminates the potential of coiled coils in the understanding of protein structure-function relationships. It introduces metal-sensitive peptide sequences for bio-sensing and photocatalysis within α-helical barrels, potentially paving the way for advancements in applications for de novo designed proteins

Prebiotic Synthesis of Inorganic Pyrophosphate (PPi) Driven by a Geochemical Redox Reaction

In all extant organisms adenosine triphosphate (ATP) is the energy carrier and transmitter of free energy from exergonic to endergonic processes. Since ATP is a complex molecule, it was unlikely available on prebiotic Earth. Proposed pyrophosphate (PPi) with energy-rich phosphoanhydride bond was considered as plausible energy-source on early Earth. In this thesis, a synthesis of PPi under prebiotic conditions is indicated by condensation of orthophosphate (Pi) in aqueous solution. Furthermore, attempts were made towards synthesizing a peptidic minimal model of the active site A-Cluster in acetyl CoA synthase (ACS), which is a key enzyme in combination with carbon monoxide dehydrogenase (CODH) in ancient WOOD-LJUNGDAHL pathway.2021-10-2