Physical Sciences and Mathematics Commons^™

Molecular-Level Studies Of Nanopatterned Biomolecules With Atomic Force Microscopy, Ashley R. Walker

LSU Doctoral Dissertations

Atomic force microscopy (AFM) is an analytical technique in which a tipped probe is gently scanned across the surface in a raster pattern to generate digital images of a sample at the nanoscale. The AFM instrument has three general operational modes, which are contact, non-contact and tapping-mode, that can be used to examine materials at the atomic level. Single-molecular details of biological molecules and other soft organic materials can be captured with minimal denaturation in either ambient or liquid environments when using tapping-mode AFM. In tapping-mode, the probe is driven to oscillate vertically while the tip is scanned across the …

A Bio-Tribocorrosion Comparison Between Additively Manufactured And Forged Ti6al4v Parts, Sina Matin, Sahar Toorandaz, Saman Nikpour, Hamidreza Abdolvand, Eshan Toyserkani, Yolanda S. Hedberg

Chemistry Publications

Ti6Al4V has been used widely as a biomedical alloy and is increasingly manufactured by additive manufacturing due to customized shapes. As implant material, it is frequently exposed to both friction and corrosive environments. This study investigates the effect of the fabrication process (laser powder bed fusion and forging) on the tribocorrosion behavior of Ti6Al4V in various environments including diluted hydrochloric acid to simulate the acidic environment in a crevice (HCl), phosphate-buffered saline (pH 7.3) with 10 g/L bovine serum albumin (PBS+BSA), and PBS+BSA with 30 mM H2O2. While the presence of BSA hindered the repassivation (reforming of the protective passive …

The Influence Of Allostery Governing The Changes In Protein Dynamics Upon Substitution, Joseph Hess

All Dissertations

The focus of this research is to investigate the effects of allostery on the function/activity of an enzyme, human immunodeficiency virus type 1 (HIV-1) protease, using well-defined statistical analyses of the dynamic changes of the protein and variants with unique single point substitutions ¹. The experimental data¹ evaluated here only characterized HIV-1 protease with one of its potential target substrates. Probing the dynamic interactions of the residues of an enzyme and its variants can offer insight of the developmental importance for allosteric signaling and their connection to a protein’s function. The realignment of the secondary structure elements can …

Structural Engineering Of Thermostable Fluorescent Proteins Tgp-E And Ytp-E And Crystal Structure Of Tgp-E, Matthew Ryan Anderson

MSU Graduate Theses

Thermostable fluorescent proteins, such as thermal green protein (TGP) and yellow thermal protein (YTP), could be used as biosensors to monitor cellular activity and as a fusion tag to monitor a protein of interest. The use of fluorescent proteins can sometimes be limited in certain organelles with low pH and in thermophilic organisms. This research aims to improve the thermal stability of TGP and YTP. TGP was created from a synthetically derived eCGP123 protein to improve solubility by substituting residues on the positively charged b- barrel surface with negatively charged glutamate (E) at Los Alamos National Lab. YTP was developed …

Fortilin Interacts With Tgf-Β1 And Prevents Tgf-Β Receptor Activation, Decha Pinkaew, Erik Martinez-Hackert, Wei Jia, Matthew D. King, Fei Miao, Nicole R. Enger, Runglawan Silakit, Kota Ramana, Shi-You Chen, Ken Fujise

Chemistry and Biochemistry Faculty Publications and Presentations

Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-β1 and prevents it from activating the TGF-β1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-β1 and its isoforms. The modified ELISA assay shows that TGF-β1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-β1. The SPR analysis also reveals that fortilin and the TGF-β receptor II …

Mechanisms And Applications Of Improved Protein Analysis By Desorption Electrospray Ionization Mass Spectrometry (Desi-Ms), Roshan Javanshad

Dissertations

Electrospray ionization mass spectrometry (ESI-MS) is a soft ionization technique that allows detection of macromolecules, such as intact proteins, by the formation of multiply charged ions from solutions. Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient ionization technique that directly samples analyte from a surface during ESI-MS analysis. Although DESI-MS is highly accomplished at the analyses of metabolites, lipids, and other small molecules, it is far more limited when it comes to protein analysis. While most of the field in ambient ionization MS has moved towards primarily applications, our approach has been to explore the use of DESI-MS and …

The Ion Pair Thermal Model Of Maldi Ms, Shiyue Fang

Michigan Tech Publications

The ion pair thermal model for MALDI MS is described. Key elements of the model include thermal desorption and ionization, strong tendency to neutralization via ion pair formation and proton transfer in the gas phase, thermal equilibrium, overall charge neutral plume, and thermal energy assisted free ion generation via ion pair separation by ion extraction potential. The quantities of ions in the solid sample and in the gaseous plume are estimated. Ion yields of different classes of molecules including peptides, nucleic acids, permanent salts and neutral molecules are estimated at the macroscale and single ion pair levels. The estimated ion …

Computational Algorithms For Predicting Membrane Protein Assembly From Angstrom To Micron Scale, Nandhini Rajagopal

Dissertations - ALL

Biological barriers in the human body are one of the most crucial interfaces perfected through evolution for diverse and unique functions. Of the wide range of barriers, the paracellular protein interfaces of epithelial and endothelial cells called tight junctions with high molecular specificities are vital for homeostasis and to maintain proper health. While the breakdown of these barriers is associated with serious pathological consequences, their intact presence also poses a challenge to effective delivery of therapeutic drugs. Complimenting a rigorous combination of in vitro and in vivo approaches to establishing the fundamental biological construct, in addition to elucidating pathological implications …

Biophysical Characterization Of The Par-4 Tumor Suppressor: Evidence Of Structure Outside The Coiled Coil Domain And Interactions With Platinum Chemotherapeutics, Andrea Megan Clark

Chemistry & Biochemistry Theses & Dissertations

Prostate apoptosis response-4 (Par-4) is an apoptosis-inducing tumor suppressor protein. Full-length Par-4 has previously been shown to be a predominantly intrinsically disordered protein (IDP) under neutral conditions, with significant regular secondary structure evident only within the C-terminal coiled coil domain. However, IDPs can gain ordered structure through the process of induced folding, which often occurs under non-neutral conditions. Previous work has shown that the Par-4 leucine zipper, which is a subset of the C-terminal coiled coil domain, is disordered under neutral conditions, but forms a dimeric coiled coil at acidic pH. Increase in ionic strength was also shown to increase …

Enhancing Plasmonic Nanomaterials: Colorimetric Sensing And Sers, John Crockett

WWU Graduate School Collection

Nanomaterials, materials with at least one dimension on the nanoscale have become an area of extreme scientific interest due to their many unique properties with applications in catalysis, optics, and sensing, just to name a few. Metal nanoparticles are particularly interesting because of the interactions between light and surface electrons in the metal’s conduction band, called localized surface plasmons. In anisotropic metal nanoparticles these plasmons are especially exciting due to the highly responsive quality of the plasmonic resonance associated with their varied nano dimensions. Gold nanorods and nano dendrites in particular exhibit electromagnetic effects which are specifically associated to the …

Development And Implementation Of A Novel Voltage-Free Interface For Capillary Electrophoresis-Mass Spectrometry Separations Of Proteins, Courtney J. Kristoff

Graduate Theses, Dissertations, and Problem Reports

Capillary electrophoresis-mass spectrometry is a powerful technique for high-throughput and high efficiency separations combined with structural identification. Electrospray ionization is the primary interface used to couple capillary electrophoresis to mass analyzers; however, improved designs continue to be reported. A new interfacing method based on vibrating sharp-edge spray ionization is presented in this work to overcome the challenges of decoupling applied voltages and to enhance the compatibility with separations performed at near-neutral pH. The versatility and ease of use of this ionization source is demonstrated using β-blockers, peptides, and proteins. The cationic β-blocker pindolol was injected electrokinetically and detected at concentrations …

Synthesis, Stabilization, And Modification Of Zinc Oxide Nanoparticles For Biological Applications, Allison Kimberly Freese

MSU Graduate Theses

Nanoparticles have become very useful as delivery systems in biomedicine. The nanoparticles can be layered with different compounds to produce a vessel for transport of biological materials. Specifically, gold nanoparticles layered with a reducing agent, lysozyme, and polyelectrolytes can be synthesized to transport lysozyme into a cell. However, zinc oxide nanoparticles are cheaper, biocompatible nanoparticles that can be used for the same process. Here in, zinc oxide nanoparticle conjugates were synthesized, modified, and analyzed to be used as a biological material delivery system. The zinc oxide nanoparticles were synthesized using zinc chloride and sodium hydroxide. The particles were then layered …

Structural Evidence For Consecutive Hel308-Like Modules In The Spliceosomal Atpase Brr2, L. Zhang, T. Xu, Corina Maeder, L.-O. Bud, J. Shanks, J. Nix, C. Guthrie, J. A. Pleiss, R. Zhao

Corina Maeder

Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2's helicase-like domains and domains 1-3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with …

Infrared Laser Ablation For Biomolecule Sampling, Kelin Wang

LSU Doctoral Dissertations

In this research, an infrared laser at a wavelength of 3 µm was used to ablate material from tissue sections for biomolecule analysis. Pulsed infrared (IR) irradiation of tissue with a focused laser beam efficiently removed biomolecules, such as proteins, enzymes, DNA, and RNA from tissue sections for further analysis. In a proteomics project, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to determine regions of interest (ROI) for laser ablation. The matrix was then washed off. By overlaying the MSI generated heat-map, the section was sampled using IR laser ablation and custom stage-control software. Two ROI were selected …

Sers For Protein Detection At A Single Molecule Level For Developing A New Medical Diagnostics Platform, Lamyaa Almehmadi

Legacy Theses & Dissertations (2009 - 2024)

A two-step process of protein detection at a single molecule level using Surface Enhanced Raman Spectroscopy (SERS) was developed as a new platform for medical diagnostics in this proof-of-concept study. First, a protein molecule was bound to a linker in the bulk solution and then this adduct was chemically reacted with the SERS substrate. Traut’s Reagent (TR) was used to thiolate Bovine serum albumin (BSA) in solution followed by chemical cross linking to a gold surface through a sulfhydryl group. A Glycine-TR adduct was used as a control sample to identify the protein contribution to the SER spectra. Gold SERS …

Isopeptide Ligations Catalyzed By Streptococcus Suis Sortase A, Sarah Bowersox

WWU Graduate School Collection

Chemically modified proteins are critical components of modern therapeutics and basic research. To generate non-natural protein derivatives, bacterial sortase enzymes have been effective due to their ability to catalyze selective ligations between protein targets and functional groups that are uncommon in nature. Thus far, the enzymatic approach using sortase has been limited to modifications at the termini of peptide chains. Here we describe efforts to develop a sortase-mediated strategy for the formation of isopeptide bonds at the side chains of internal lysine residues. To this end, we have identified a sortase A homolog from Streptococcus suis (SrtA_suis) that …

Investigations Into Factors Affecting The Wpd-Loop In The Protein Tyrosine Phosphatases Yoph And Ptp1b, Gwendolyn Moise

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The research in this dissertation documents connections between the primary amino acid sequence of proteins, the dynamics of proteins, and their catalytic function. This research project studied two proteins called protein-tyrosine phosphatases (PTPs): the human enzyme PTP1B, and the bacterial enzyme YopH. PTP1B is a human enzyme that down regulates the insulin receptor on the outer cellular membrane, and causes the insulin receptor to be less responsive to insulin. A deeper knowledge of how PTP1B is different from other human PTPs might be useful in designing drugs to increase insulin sensitivity in diabetics. Yersinia Pestis is the bacteria that caused …

Detrital Protein Contributes To Oyster Nutrition And Growth In The Damariscotta Estuary, Maine, Usa, Cheyenne M. Adams

Electronic Theses and Dissertations

Oyster aquaculture is an expanding industry that relies on identifying and utilizing natural estuarine conditions for the economically viable production of a filter-feeding crop. The eastern oyster, Crassostrea virginica, is the principal species currently cultured in Maine. In addition to preferentially consumed phytoplankton, various detrital complexes (non-algal and/or non-living organic matter) may provide some nutrition to C. virginica between times of phytoplankton abundance. Here I investigated the importance of detrital proteins in supporting the growth of oysters cultured in the upper Damariscotta Estuary. Oyster aquaculture in this area is highly successful and previous reports indicate that labile detrital protein …

Formulation And Characterization Of Lipogels As A Tunable Delivery Scaffold, Celia Homyak

Doctoral Dissertations

Nanomedicines within the clinic commonly utilize lipid-based scaffolds due to their aqueous assembly and non-covalent dual-guest loading capabilities. Nevertheless, poor stability in vivo and premature guest release remains a challenge with these systems. Polymeric nanomaterials are a popular alternative due to their good stability in vivo with controllable guest release. A caveat to polymeric scaffolds though, is the excess synthetic effort involved for non-covalent dual-drug encapsulation. Benefit lies in harnessing advantages of lipid and polymeric materials via lipid-polymer hybrid scaffold. Such materials have promise due to their dual-encapsulation and dual-stimuli-sensitive characteristics. The need to better understand these materials led us …

Green Chemistry Oxidative Modification Of Peptoids Utilizing Bleach And Tempo, Jesse Leland Roberts

Graduate Theses and Dissertations

Biotherapeutic drugs, derived from biological molecules such as proteins and DNA, are becoming an integral and exceptionally critical aspect of modern medicine. Compared to common pharmaceutical drugs, biotherapeutics are much larger in size and have greater target specificity, allowing them to treat many chronic diseases ranging from cancer to rheumatoid arthritis. The major issue with protein based therapeutics is that they readily undergo proteolysis, or enzymatic degradation, when administered through subcutaneous injections. Traditionally, biotherapeutic modification procedures have centered on the use of PEG derivatives. This process, called PEGylation, is unfavorable due to the increases in molecular weights of the proteins …

Bioinformatic And Biophysical Analyses Of Proteins, Jonathan Catazaro

Department of Chemistry: Dissertations, Theses, and Student Research

The prevailing dogma in structural genomics is the existence of a strong correlation between protein sequence, structure, and biological function. Proteins with high sequence similarity typically have a similar, if not the same, structure and function. In many cases this logic can fail due to distantly related proteins having very low sequence similarity, a lack of a representative structure, structural novelty, or the absence of a characterized function. Further, the paradigm fails to account for dynamics, which have a significant effect on structural stability and enzymatic efficacy.

Nuclear magnetic resonance (NMR) spectroscopy is uniquely capable of solving the structure, assisting …

Identification Of Hydrated And Dehydrated Lipids And Protein Secondary Structures In Seeds Of Cotton (Gossypium Hirsutum) Line, Diwas Kumar Silwal, Nsoki Phambu, Bharat Pokharel, Ahmad Naseer Aziz

Chemistry Faculty Research

Cottonseeds from two parents (TM-1 and 3-79) and their 17 progeny (chromosomal substitution) lines were analyzed for various secondary structures of proteins and moisture content of lipids, separately in hulls and kernels. Fourier transform infrared spectroscopy (FTIR) was used on mature seeds from Upland cotton (G. hirsutum) progeny lines and parents. Based on secondary structures of proteins and hydration levels of lipids, differences were observed among the cottonseeds. The two progeny lines – CS-B12sh and CS-B22sh retained lipid moisture content and protein secondary structures similar to both parents, while CS-B06, CS-B15sh and CS-B16 remained distinct from either parent. On the …

A Systematic Investigation Of The Effects Of Chain Length And Ionic Head Group On Perfluoroalkyl Acid Binding To Human Serum Albumin, Jake Ulrich

Honors Theses

Perfluoroalkyl acids (PFAAs) are industrial chemicals used in everyday products ranging from non-stick coatings to fire-fighting foam. PFAAs are contaminants of emerging concern (CECs) and are bioaccumulative, persistent and toxic. Unlike other CECs, PFAAs bioaccumulate in areas of high protein concentration, such as the kidneys, liver and blood; therefore, it is vital to study PFAA-protein interactions. Human Serum Albumin (HSA) is the model protein used for PFAA-protein studies because it is the most abundant protein in the human body and it binds and transports endogenous and exogenous ligands. Previously, researchers have investigated PFAA-HSA binding, but most of these studies have …

Mutagenic And Spectroscopic Investigation Of Ph Dependent Cooa Dna Binding, Brian R. Weaver

Chemistry Honors Papers

The carbon monoxide (CO) sensing heme protein, CooA, is a transcription factor which exists in several bacteria that utilize CO as an energy source. CooA positively regulates the expression of coo genes in the presence of CO such that the corresponding proteins may metabolize CO. The present studies have yielded the unexpected result that Fe(III) CooA binds DNA tightly at pH < 7, deviating from all previously reported work which indicate that CooA DNA binding is initiated only when the exogenous CO effector reacts with the Fe(II) CooA heme. This observation suggests that the disruption of one or more salt bridges upon effector binding may be a critical feature of the normal CooA activation mechanism. To test this possibility, several protein variants that eliminated a selected salt bridge for the CooA homolog from Rhodospirillum rubrum were prepared via site-directed mutagenesis. Samples of these variant proteins, which were overexpressed in Escherichia coli, were then characterized by spectroscopic methods and functional assays to investigate the impact these mutations had on CooA heme coordination …

Nanoscale Studies Of Proteins And Thin Films Using Scanning Probe Microscopy, Zachary Lane Highland

LSU Doctoral Dissertations

Nanostructures of organosilanes, thin metal films, and protein nanopatterns were prepared and analyzed with atomic force microscopy (AFM). Organosilanes with designed functional groups were used to selectively pattern green fluorescent protein at the nanoscale using protocols developed with particle lithography. Mesospheres are deposited onto a substrate to produce a surface mask. Organosilanes are deposited to form a matrix film surrounding nanopores for depositing proteins. The nanopatterns were characterized using AFM, after steps of particle lithography for directly visualizing surface changes. Studies with AFM also provide a compelling tool for teaching undergraduates to introduce concepts of nanoscience. An undergraduate laboratory was …

Expression And Purification Of Atg11: A Protein Involved In The Organization Of Autophagy, Carson Zois

Senior Honors Theses and Projects

Autophagy is a physiological process for the degradation of internal cell components to promote cell survival. This process allows the cell to maintain homeostasis, recycle damaged cellular components and helps protect against disease. Autophagy can be selective or non-selective. Non-selective, or bulk, autophagy occurs during times of cellular starvation and results in non-specific degradation of cytoplasmic materials. Selective autophagy targets specific components within the cytoplasm for degradation, and is always active. In both forms of autophagy, components are degraded when a double-membraned structure, called the autophagosome, encapsulates the target cargo and is then recruited to the lysosome in mammalian cells, …

Interaction Of Spliceosomal U2 Snrnp Protein P14 With Its Branch Site Rna Target, William Perea Vargas

Dissertations, Theses, and Capstone Projects

Newly transcribed precursor messenger RNA (pre-mRNA) molecules contain coding sequences (exons) interspersed with non-coding intervening sequences (introns). These introns must be removed in order to generate a continuous coding sequence prior to translation of the message into protein. The mechanism through which these introns are removed is known as pre-mRNA splicing, a two-step reaction catalyzed be a large macromolecular machine, the spliceosome, located in the nucleus of eukaryotic cells. The spliceosome is a protein-directed ribozyme composed of small nuclear RNAs (snRNA) and hundreds of proteins that assemble in a very dynamic process. One of these snRNAs, the U2 snRNA, is …

Purification And Preparation Of Intrinsically Disordered Proteins For Nmr Spectroscopy, Kyle Chamberlain

Open Access Theses

NMR spectroscopy has recently become a promising field for protein characterization and dynamic studies. As the technology and pulse sequences improve for tracking proteins, a greater demand for developing effective purification protocols to produce NMR grade protein samples have arisen. This thesis explores two proteins: histone H4 tail and Mucin 1; Two very different proteins that require different methods of expression and purification to achieve a high enough yield for NMR analysis. H4 is a water soluble protein that weighs ~2.7 kDa, and has no extinction coefficient. Since the protein is too small for many methods of expression and purification, …

Supramolecular Strategies For The Generation Of Nanoparticle Assemblies And Biomolecular Thin Films, Bradley P. Duncan

Doctoral Dissertations

The conceptual framework of supramolecular chemistry elucidates a powerful set of strategies for chemists to generate functional nanomaterials based on intermolecular forces. My research focused on tuning the molecular interactions of nanoscale components to create larger structures with enhanced properties. In one approach, I developed and optimized an additive-free, nanoimprint lithography-based methodology to generate stable thin films from a variety of proteins. The generalized process retains intrinsic properties of the protein as demonstrated by selective cellular adhesion. The heat and pressure of the nanoimprinting process induces slight structural reorganization of the peptide side chains to yield highly stable films held …

Rcd+: Fast Loop Modeling Server, José R. López-Blanco, Alejandro J. Canosa-Valis, Yaohang Li, Pablo Chacón

Computer Science Faculty Publications

Modeling loops is a critical and challenging step in protein modeling and prediction. We have developed a quick online service (http://rcd.chaconlab.org) for ab initio loop modeling combining a coarse-grained conformational search with a full-atom refinement. Our original Random Coordinate Descent (RCD) loop closure algorithm has been greatly improved to enrich the sampling distribution towards near-native conformations. These improvements include a new workflow optimization, MPI-parallelization and fast backbone angle sampling based on neighbor-dependent Ramachandran probability distributions. The server starts by efficiently searching the vast conformational space from only the loop sequence information and the environment atomic coordinates. The generated closed loop …