Protein Redesign and De Novo Design Approaches to Create Artificial Nickel-Dependent Metalloenzymes

Energy crisis, energy demand and climate change are the major challenges of this century. As we face these challenges, it’s important that we depend on alternate energy forms like renewable energy sources and at the same devise ways to tackle the greenhouse effect. Therefore, it is important to move towards a carbon neutral energy economy where we can convert atmospheric carbon dioxide back into organic carbon and depend on clean energy sources like Hydrogen. These energy concerns can be tackled well if we understand the role of enzymes responsible for various biological reactions.Metals play a variety of unexpected roles in biological systems and are essential to the existence of life. Enzymes with metal bound active site that catalyzes reactions are called as metalloenzymes. We are interested in learning about two such complex metalloenzymes, Hydrogenases and Acetyl Coenzyme A Synthases (ACS) which is part of an enzyme complex CODH/ACS. Hydrogenases can catalyze the interconversion of protons into H2 and vice versa at a very low overpotential (\u3c100 \u3emV). The CODH/ACS enzymes together can reduce CO2 into CO and couple two carbon compounds along with Coenzyme A (CoA) to make an important metabolite Acetyl-CoA. However, the complex nature of these enzymes along with poor yield and oxygen sensitivity restrict their study under normal laboratory conditions. To understand the structural and functional details of these enzymes and to use them for environmental purposes, we use protein engineering and de novo protein design approaches to make small biological model systems called Artificial Metalloenzymes (ArMs). The ArMs are often designed based on the metal active center of the metalloenzymes. We use various spectroscopic techniques to understand metal binding and the hidden mechanism of the catalytic center and electrochemical and photochemical techniques to evaluate the catalytic efficiencies of these systems. This dissertation focuses on the protein reengineering and de novo design approaches to build Artificial Hydrogenases (ArHs) and Artificial Acetyl Coenzyme A Synthases (Ar-ACSs). In, chapter 3, we have redesigned a copper storage protein into a nickel binding protein (NBP) and proved that Ni-NBP is an ArH through electrochemical and photochemical techniques. In chapter 4, we studied the oxygen sensitivity of the NBP through electrochemistry and found that it catalyzes the complete 4e- oxygen reduction reaction at low pH. In chapter 5, we designed a de novo peptide with NiS4 at the active site and studied the incorporation of a synthetic Fe complex to build a bimetallic active site similar to the [NiFe] hydrogenase. In chapter 6, we designed trimeric peptide assembly based on de novo design that makes a NiS3 active site, which binds and couples two carbon compounds (CO and -CH3) making it an Ar ACS enzyme. In chapter 7, we pursued the original research proposal on redesigning a ferredoxin scaffold to build the active center of ACS enzyme and studied the metal and ligand binding through various techniques

A De Novo Designed Trimeric Metalloprotein as a Ni_p Model of the Acetyl-CoA Synthase

We present a Nip site model of acetyl coenzyme-A synthase (ACS) within a de novo-designed trimer peptide that self-assembles to produce a homoleptic Ni(Cys)3 binding motif. Spectroscopic and kinetic studies of ligand binding demonstrate that Ni binding stabilizes the peptide assembly and produces a terminal NiI-CO complex. When the CO-bound state is reacted with a methyl donor, a new species is quickly produced with new spectral features. While the metal-bound CO is albeit unactivated, the presence of the methyl donor produces an activated metal-CO complex. Selective outer sphere steric modifications demonstrate that the physical properties of the ligand-bound states are altered differently depending on the location of the steric modification above or below the Ni site

Annotation of the Zebrafish Genome through an Integrated Transcriptomic and Proteomic Analysis

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes

A draft map of the human proteome

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease. © 2014 Macmillan Publishers Limited