Enhanced Ion Activation and Dissociation of Intact Proteins in Native Mass Spectrometry Based Top-Down Proteomics

Honors (Bachelor's)ChemistryUniversity of Michiganhttps://deepblue.lib.umich.edu/bitstream/2027.42/147366/1/keatingm.pd

Fluorescence Correlation Spectroscopic Study of Serpin Depolymerization by Computationally Designed Peptides

Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found

Comprehensive Overview of Causative Agents of Alzheimer\u27s Disease: Tau Protein and Amyloid Betas With Their Biochemical Pathways and Proposed Treatments Including Cost Analysis

Alzheimer’s is a neurodegenerative disease found within the brain, interfering with neuron function, eventually leading to widespread atrophy. The disease effects millions of Americans with neurofibrillary tangles and amyloid beta plaques, both protein deposits with unclear causes. The goal for this thesis was not only to understand how these proteins form but how to safely interfere with their production. This was completed by a comprehensive overview of the form of the buildups and their precursors, tau proteins and amyloid beta precursor protein, respectively. An emphasis was put on the molecular biology and genetic causes of the amyloids rather than the large-scale symptomatic results. With a systematic review of scientific results and findings, a hypothesis was formed on how these proteins aggregate and techniques that cannot only treat but prevent the proteins, along with an analysis of treatment costs

Theoretical Circular Dichroism Of Lysozyme, Myoglobin And Collagen And Experimental Circular Dichroism Of Myoglobin And Pea Lectin

The need to understand protein structure and interaction is ever-growing and this has led to scientist and investigators utilizing numerous different techniques in order to obtain substantial insights and explanation to these structures and their interactions. Circular dichroism (CD), which is one of these techniques, is a powerful structural biology technique used to study protein and nucleic acid structures and their dynamics. This technique is important because it identifies the secondary, tertiary and even quaternary structures in proteins and can be used to study folding patterns in proteins. Theoretical and experimental methods are used to better understand and teach the phenomenon of circular dichroism. First, molecular mechanics allows for the energy calculations of different conformations in large molecules like peptides and proteins. Theoretical CD via the dipole interaction model (DInaMo) is used to relate the structural nature of peptides and proteins to the experimental CD observed. Minimization was done on lysozyme, myoglobin and collagen, using the molecular modeling software package, Insight®II to obtain minimum energy structures suitable for CD calculations. Molecular dynamics simulations were performed in water at 300K to create an ensemble of conformations. The program CDCALC was used to predict the CD spectrum of the proteins for comparison with experiment. The output from CDCALC was analyzed using OriginPro Version 7.5 and the analyzed data reported as plots with data from synchrotron radiation circular dichroism (SRCD). Theoretical CD plots showed agreement with SRCD for location, sign, and bandwidths of the peaks. Experimental CD spectra of horse heart myoglobin and pea lectin were measured on a JASCO spectropolarimeter and compared to that obtained from the Protein Circular Dichroism Data Bank (PCDDB) in the development of a physical chemistry laboratory exercise to teach secondary structure analysis

Investigating the Structure of the Papain-Inhibitor Complex using SPR and NMR

Cysteine proteases (CPs) are enzymes with a nucleophilic thiol in their active sites. Inhibitors of cysteine proteases (ICPs) occur naturally in bacterial pathogens and some protozoa. In parasites, ICPs are often virulence factors, contributing to the formation and survival of amastigotes within host cells. These amastigotes have higher CP activity, therefore making both ICPs and CPs potential drug targets. Despite great genetic variability, ICPs contain highly conserved structural features, including a series of defined loops that play a significant role in binding CPs. Papain, a CP from Carica papaya, complexes with ICP from Leishmania mexicana. Although the individual 3-D structures of ICP and papain have been determined, as of this work, the structure of the papain-ICP complex has only been predicted, not solved. This research details the development of a technique for determining quaternary structure of the papain-ICP complex using paramagnetic relaxation enhancement NMR (PRE-NMR). A paramagnetic tag (MTSL) was added to various cysteine-mutants of ICP to measure distances to reductively 13C-methylated papain. The modification of ICP with MTSL was quantified using EPR, and the effects of labeling on the binding kinetics of papain and ICP were determined using SPR. 13C-methyl peak perturbations due to PRE were observed when papain was bound to spin-labeled E102C-ICP and K27C-ICP. Intermolecular distances were predicted using modeling software and a working model of the complex was created. Data from additional mutants will help to further determine complex structure and perfect the model.The penultimate chapter of this dissertation includes work towards the development of a method for studying protein-protein interactions using atomic force microscopy. Papain-ICP was used as a model system, with the intention to apply this method to the study of another system: filamentous actin (f-actin) and the actin-binding domain of abelson tyrosine-protein kinase (ABL2-FABD). The creation of nanopores on an AFM sensor chip surface was successful. ICP monomers bound selectively into the pores. Attempts to form the papain-ICP complex on the chip surface were unsuccessful, and future work is needed to perfect this method. The final chapter of this dissertation is a literature review outlining previous work in this area

A threading approach to protein structure prediction: studies on TNF-like molecules, Rev proteins, and protein kinases

The main focus of this dissertation is the application of the threading approach to specific biological problems. The threading scheme developed in our group targets incorporating important structural features necessary for detecting structural similarity between the target sequence and the template structure. This enables us to use our threading method to solve problems for which sequence-based methods are not very much useful. We applied our threading method to predict the three-dimensional structures of lentivirus (EIAV, HIV-1, FIV, SIV) Rev proteins. Predicted structures of Rev proteins suggest that they share a structural similarity among themselves (four-helix bundle). Also, the threading approach has been utilized for screening for potential TNF-like molecules in Arabidopsis. The threading approach identified 35 potential TNF-like proteins in Arabidopsis, six of which are particularly interesting to be tested for the receptor kinase ligand activity. Threading method has also been used to identify potentially new protein kinases, which are not included in the protein kinase data base of C. elegans and Arabidopis. We identified eleven potentially new protein kinases and an additional protein worth investigating for protein kinase activity in C. elegans. Further, we identified ten potentially new protein kinases and additional four proteins worth investigating for the protein kinase activity in Arabidopsis

Data Enrichment for Data Mining Applied to Bioinformatics and Cheminformatics Domains

Problemas cada vez mais complexos estão a ser tratados na àrea das ciências da vida. A aquisição de todos os dados que possam estar relacionados com o problema em questão é primordial. Igualmente importante é saber como os dados estão relacionados uns com os outros e com o próprio problema. Por outro lado, existem grandes quantidades de dados e informações disponíveis na Web. Os investigadores já estão a utilizar Data Mining e Machine Learning como ferramentas valiosas nas suas investigações, embora o procedimento habitual seja procurar a informação baseada nos modelos indutivos. Até agora, apesar dos grandes sucessos já alcançados com a utilização de Data Mining e Machine Learning, não é fácil integrar esta vasta quantidade de informação disponível no processo indutivo, com algoritmos proposicionais. A nossa principal motivação é abordar o problema da integração de informação de domínio no processo indutivo de técnicas proposicionais de Data Mining e Machine Learning, enriquecendo os dados de treino a serem utilizados em sistemas de programação de lógica indutiva. Os algoritmos proposicionais de Machine Learning são muito dependentes dos atributos dos dados. Ainda é difícil identificar quais os atributos mais adequados para uma determinada tarefa na investigação. É também difícil extrair informação relevante da enorme quantidade de dados disponíveis. Vamos concentrar os dados disponíveis, derivar características que os algoritmos de ILP podem utilizar para induzir descrições, resolvendo os problemas. Estamos a criar uma plataforma web para obter informação relevante para problemas de Bioinformática (particularmente Genómica) e Quimioinformática. Esta vai buscar os dados a repositórios públicos de dados genómicos, proteicos e químicos. Após o enriquecimento dos dados, sistemas Prolog utilizam programação lógica indutiva para induzir regras e resolver casos específicos de Bioinformática e Cheminformática. Para avaliar o impacto do enriquecimento dos dados com ILP, comparamos com os resultados obtidos na resolução dos mesmos casos utilizando algoritmos proposicionais.Increasingly more complex problems are being addressed in life sciences. Acquiring all the data that may be related to the problem in question is paramount. Equally important is to know how the data is related to each other and to the problem itself. On the other hand, there are large amounts of data and information available on the Web. Researchers are already using Data Mining and Machine Learning as a valuable tool in their researches, albeit the usual procedure is to look for the information based on induction models. So far, despite the great successes already achieved using Data Mining and Machine Learning, it is not easy to integrate this vast amount of available information in the inductive process with propositional algorithms. Our main motivation is to address the problem of integrating domain information into the inductive process of propositional Data Mining and Machine Learning techniques by enriching the training data to be used in inductive logic programming systems. The algorithms of propositional machine learning are very dependent on data attributes. It still is hard to identify which attributes are more suitable for a particular task in the research. It is also hard to extract relevant information from the enormous quantity of data available. We will concentrate the available data, derive features that ILP algorithms can use to induce descriptions, solving the problems. We are creating a web platform to obtain relevant bioinformatics (particularly Genomics) and Cheminformatics problems. It fetches the data from public repositories with genomics, protein and chemical data. After the data enrichment, Prolog systems use inductive logic programming to induce rules and solve specific Bioinformatics and Cheminformatics case studies. To assess the impact of the data enrichment with ILP, we compare with the results obtained solving the same cases using propositional algorithms

Characterization of autoclaved flaxseed as feed for ruminants using conventional and mid-IR spectroscopic based approaches

The objectives of this study were to investigate the effects of autoclave heating on the rumen protein degradation characteristics of flaxseed (Linum usitatissimum, cv. Vimy), and to compare them to differences in diffuse reflectance infrared Fourier transform (DRIFT) and Synchrotron based Fourier transform infrared microspectroscopy (S-FTIR) measurements of the protein alpha-helix to beta-sheet ratios. Hierarchical cluster analysis (CLA) and principal components analysis (PCA) were also conducted to identify differences in the DRIFT spectra. Flaxseed samples were kept raw for control or autoclaved in batches at 120°C for 20, 40 or 60 min for treatments 1, 2 and 3, respectively. The rumen degradation kinetics of protein were measured along with the protein sub-fractions of the Cornell net carbohydrate and protein system (CNCPS), and chemical composition. Intestinal digestibility was determined using the three-step procedure outlined by Calsamiglia and Stern (1995). Protein supply to the small intestine was determined using the NRC (2001) and DVE/OEB models. The results showed that heating increased dry matter (DM) and ether extract (EE) content, while reducing neutral detergent fibre (NDF) and acid detergent fibre (ADF), with little numerical difference between the three treatments. Soluble crude protein (SCP) also decreased upon autoclaving with concomitant increases in non-protein nitrogen (NPN), neutral detergent insoluble nitrogen (NDIN) and acid detergent insoluble nitrogen (ADIN). The CNCPS protein sub-fractions with the greatest changes were the buffer-soluble true protein fraction (PB1) and the fraction representing buffer-insoluble true protein which is not bound to NDF (PB2) showing dramatic increases, indicating a decrease in the overall protein degradability. In situexperiments showed a reduction in effective degradable dry matter (EDDM) as well as a reduction in effective degradable crude protein (EDCP) without significant differences between the treatments. Intestinal digestibility of protein as estimated by the three-step procedure showed no changes upon autoclaving. Modeling results, with flaxseed as the only feed source, for absorbable ruminally-undegraded feed protein in the intestines using both the NRC (2001) and DVE/OEB systems showed increases as a consequence of the autoclave treatments but again there were no differences between the treatments. The degraded protein balance results showed for both the NRC (2001) and DVE/OEB models that both were decreased upon autoclave treatment. However, the values for the NRC (2001) model suggested a potential nitrogen (N) deficiency and, therefore potentially impaired microbial crude protein (MCP) production, whereas the values for the DVE/OEB system showed potential N excess and, therefore, possible loss from the rumen. DRIFT analysis of protein secondary structure ratios showed a decrease in the alpha-helix to beta-sheet ratio for the whole seed, whereas results from S-FTIR spot data for cotyledon tissue showed autoclaving had the opposite effect on the ratio. CLA and PCA were successfully used to make distinctions between the different treatment spectra and showed enhanced sensitivity upon selection of a smaller spectral window to include only the amide I and II portion of the IR spectrum. The results failed to demonstrate any differences between the autoclave treatments used in this study, and showed that autoclaving generally decreased effectively ruminal degradability of flaxseed protein. The results further indicated that autoclaving had a significant enough effect on the flaxseed to permit identification of the altered alpha-helix to beta-sheet ratio with the mid-IR spectrum, as well as differentiation between the treatments using PCA and CLA. PCA and CLA results suggest that mid-IR spectral methods are more sensitive than traditional methods when used to identify differences between the heat treatments

Studies on the unfolding and refolding of multi-subunit proteins

Studies on the unfolding and refolding of multi-subunit proteins have been carried out and the results obtained for translocated proteins have been compared where possible with those obtained for non-translocated proteins. This comparison should indicate the way in which translocation affects the folding and assembly of such proteins. The enzymes studied were the glutamate dehydrogenases from bovine liver, baker's yeast and Clostridium symbiosum: the cytoplasmic and mitochondrial aspartate aminotransferase isoenzymes from pig heart and citrate synthase from pig heart. The unfolding of the enzymes by guanidinium chloride (GdnHCl) was studied by monitoring loss of catalytic activity and changes in structure by fluorescence, circular dichroism and exposure of reactive thiol groups. It was found that loss of enzyme activity occurs at lower concentrations of GdnHCl than any major changes in the structure. The refolding of the enzymes was studied by measuring the regain of catalytic activity on dilution of the GdnHCl. It was found that only the cytoplasmic aspartate aminotransferase enzyme was able to regain activity from the unfolded state. In comparison, the translocated proteins studied bovine liver glutamate dehydrogenase, mitochondrial aspartate aminotransferase and citrate synthase could not be refolded after denaturation. This inability of translocated proteins to refold from their isolated, unfolded subunits implies that other factors are involved i the folding and assembly of translocated proteins in vivo. It is possible that chaperone proteins nay be involved in this process

Biomimetic models for redox enzyme systems

Supramolecular chemistry involves the study of noncovalent interactions that take place between molecules. A supramolecule or host-guest complex is formed when a noncovalent binding or complexation event occurs between two such molecules. Hydrogen bonds, electrostatics, pi-stacking, hydrophobic effects, solvatophobic effects and van der Waals forces are all types of noncovalent interactions. Biological systems have provided much of the inspiration for the development of supramolecular chemistry, and many synthetic supramolecular systems have been designed to mimic biological and enzymatic processes. Biomimetic modelling involves the synthesis of compounds containing similar functional groups to that of the specific enzyme’s protein and cofactor. Subsequent analysis using chemical, physical or computational techniques can be used to gain a better understanding of the interactions taking place. This study involves the investigation of various biomimetic redox enzyme systems. Firstly, model systems containing the 1- and 5-deazaflavin cofactor have been synthesised and studied to probe how their redox behaviour compares to that of riboflavin in a supramolecular environment using physical, electrochemical and computational techniques. Secondly, this study has focussed on the flavin cofactor but has expanded upon what factors influence its redox behaviour, and ability to noncovalently interact with other molecules, by examining how the presence of different dendritic architectures can affect its redox properties and noncovalent behaviour. A series of dendrons have been synthesised and studied that have a water-soluble dendron architecture attached to the flavin moiety, as well as a series of dendrons with branching designed to encapsulate the flavin unit. Finally, a biomimetic model of the pyrroloquinoline quinone cofactor has also been synthesised and studies carried out to investigate its redox behaviour in a supramolecular environment, and ability to noncovalently interact with other molecules. The results of this study will hopefully contribute significantly to the body of chemical research in the area of supramolecular chemistry and biomimetics. Of particular interest will be the results from the flavin-based dendron research, as the prospect of purpose-built synthetic enzymes, designed and synthesised for whatever role is required, would surely be of great significance