Deep Ultraviolet Resonance Raman Spectroscopy of Membrane Proteins
Author | : Christopher Halsey |
Publisher | : |
Total Pages | : 95 |
Release | : 2012 |
Genre | : Electronic Dissertations |
ISBN | : |
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Author | : Christopher Halsey |
Publisher | : |
Total Pages | : 95 |
Release | : 2012 |
Genre | : Electronic Dissertations |
ISBN | : |
Author | : Mia C. Brown |
Publisher | : |
Total Pages | : 94 |
Release | : 2015 |
Genre | : |
ISBN | : |
Membrane proteins perform a variety of functions within our cells. They transport nutrients and waste across the lipid barrier, transmit signals from one part of the body to another, and run our immune system. However, despite their ubiquitous and vital presence in all organisms, relatively little is known about this class of proteins compared to their soluble counterparts. In this work I have set out to use deep UV resonance Raman (DUVRR) spectroscopy to characterize structural and environmental transitions of proteins and applied the results to studies involving intramembrane proteases and their substrates. DUVRR has been used extensively to observe protein secondary structure. This work contains the results of three main studies I have conducted during my graduate career. In the first I show results from the first experiment to look simultaneously at both secondary and tertiary structure as a protein transitions from a molten globule to ordered state. In the second study tracks the structural and environmental changes of a small peptide as it transitions from a soluble, disordered state through two membrane-bound, structurally ordered states. I was then able to apply the findings of these experiments to study intramembrane proteolysis, wherein I describe substrate characteristics and their interactions with proteases.
Author | : Anahita Zare |
Publisher | : |
Total Pages | : 109 |
Release | : 2017 |
Genre | : |
ISBN | : |
Membrane protein function and structure determination is vital for pharmaceutical development and disease prevention. Despite the various methods of protein structure elucidation in use, a need still exists for techniques that are of adequate resolution, rapid, inexpensive, and compatible with the membrane environment of these proteins. Deep UV resonance Raman (dUVRR) spectroscopy is an emerging structurally sensitive spectroscopic technique for analyzing membrane protein structure. The backbone amide modes are resonance enhanced in dUVRR spectra while the membrane's lipid features are not, resulting in strong membrane protein spectral features in near native environments. In order to better define dUVRR spectral features of membrane proteins, a series of model helical peptides, poly(LA)7, were designed. By substituting select residues of the transmembrane region of poly(LA)7, the propensity of helical structure within a membrane has also been studied. Using these model membrane peptides in increasingly dehydrated environments (aqueous, surfactant, and bilayer), hydration depended changes in the spectra are characterized.
Author | : Katheryn Marie Sanchez |
Publisher | : |
Total Pages | : 230 |
Release | : 2010 |
Genre | : |
ISBN | : 9781109691238 |
This dissertation focuses on the folding dynamics of a bacterial membrane protein, Outer Membrane Protein A (OmpA), using fluorescence and Ultraviolet resonance Raman spectroscopy. Our model [Beta]-barrel membrane protein, OmpA, contains five native anchoring Tryptophan residues. The spectroscopic properties of trp residues are highly sensitive to the local environment, making it an ideal probe for membrane protein folding studies. Utilizing trp fluorescence, refolding studies were performed on single trp mutants of OmpA to determine the thermodynamic stability of these trp mutants. The important noncovalent interactions that promote stability in OmpA are pairwise aromatic interactions and hydrogen bonds with the N1H moiety of trp. Refolding studies were also performed on truncated single-trp mutants, in which the soluble domain of the protein was removed. These studies resulted in increased stability relative to the full-length protein and suggest the absence of the soluble domain may destabilize the unfolded transmembrane domain. Ultraviolet resonance Raman spectroscopy (UVRR) is a powerful vibrational technique that can selectively probe different biological chromophores depending on excitation wavelength. Excitation wavelength dependence studies were performed on OmpA using wavelengths from 206.5 nm - 236.5 nm. This study determined an optimal excitation wavelength of 228-nm to selectively enhance signal from trp residues in OmpA. Additionally, UVRR was used to monitor changes in trp environmental hydrophobicity, hydrogen bonding, and dihedral torsion angle in different conformations of OmpA. The first UVRR spectra were collected of OmpA in a highly scattering environment and show differences in folded and unfolded conformations of the protein, showing the applicability of this technique to study membrane protein folding. UVRR spectra were collected of trp mutants of OmpA at different time points in the folding/insertion process to determine the types of noncovalent interactions with trp residues, and the folding timescales these interactions occur. Our results indicate noncovalent interactions start to form within the first 20 minutes after initiation of folding into DMPC vesicles and continue to show subtle changes over the course of the folding process. Additionally, there is evidence for interactions between trp residues and lipids, inter-residue hydrogen bonding, and amino-aromatic interactions.
Author | : |
Publisher | : Academic Press |
Total Pages | : 496 |
Release | : 2017-01-05 |
Genre | : Science |
ISBN | : 0128122145 |
Enzymology at the Membrane Interface: Intramembrane Proteases, Volume 584, the latest release in the Methods in Enzymology series, covers a subset of enzymes that work in the environment of the biological cell membrane. This field, called interfacial enzymology, involves a special series of experimental approaches for the isolation and study of these enzymes. Covers a subset of enzymes that work in the environment of the biological cell membrane Offers a series of experimental approaches for the isolation and study of enzymes
Author | : Tapan K. Das |
Publisher | : John Wiley & Sons |
Total Pages | : 380 |
Release | : 2014-04-28 |
Genre | : Medical |
ISBN | : 0470938439 |
With a focus on practical applications of biophysical techniques, this book links fundamental biophysics to the process of biopharmaceutical development. • Helps formulation and analytical scientists in pharma and biotech better understand and use biophysical methods • Chapters organized according to the sequential nature of the drug development process • Helps formulation, analytical, and bioanalytical scientists in pharma and biotech better understand and usestrengths and limitations of biophysical methods • Explains how to use biophysical methods, the information obtained, and what needs to be presented in a regulatory filing, assess impact on quality and immunogenicity • With a focus on practical applications of biophysical techniques, this book links fundamental biophysics to the process of biopharmaceutical development.
Author | : Olayinka Oshokoya |
Publisher | : |
Total Pages | : 132 |
Release | : 2015 |
Genre | : |
ISBN | : |
Determination of protein secondary structure ([alpha]-helical, [beta]-sheet, and disordered motifs) has become an area of great importance in biochemistry and biophysics as protein secondary structure is directly related to protein function and protein related diseases. While NMR and x-ray crystallography can predict placement of each atom in proteins to within an angstrom, optical methods (CD, Raman, IR) are the preferred techniques for rapid evaluation of protein secondary structure content. Such techniques require calibration data to predict unknown protein secondary structure content where accuracy may be improved with the application of multivariate analysis. We compare protein secondary structure predictions obtained from multivariate analysis of ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopic data using classical and partial least squares, and multivariate curve resolution-alternating least squares is made. Based on this analysis, the suggested best approach to rapid and accurate secondary structure determination is a combination of both CD and UVRR spectroscopy.
Author | : John Simpson |
Publisher | : |
Total Pages | : 101 |
Release | : 2009 |
Genre | : Chemometrics |
ISBN | : |
Ultra violet resonance Raman (UVRR) is a powerful spectroscopic technique for determining the secondary structural content of proteins in solution. However, the analyses of UVRR spectra can be problematic due to the difficulty of determining the pure secondary structure Raman spectra. The use of multi-excitation datasets can help to alleviate the difficulty in determining the pure secondary structure Raman spectra, but due to increasing spectral resolution as excitation wavelength increases, multi-excitation datasets are notoriously difficult to align spectral features. In addition, the subtraction of the water band can be difficult when relying on an internal intensity standard. To address these difficulties we demonstrate the use of a series of chemometric methods. To determine the pure secondary structure Raman spectra, we demonstrate the use of multivariate curve resolution using the alternating least squares algorithm (MCRALS) with a multi-excitation data set. To alleviate mis-alignment in multi-excitation data, we demonstrate the use of correlation optimized warping (COW). We also propose a new water band subtraction method which will reliably determine the water band concentration and remove it, without an over subtraction. Finally, we demonstrate the use of parallel factor analysis (PARAFAC) for the characterization of the behavior of the flavonoid quercetin in solution with the protein bovine serum albumin.
Author | : Fabio Della Sala |
Publisher | : CRC Press |
Total Pages | : 500 |
Release | : 2013-08-13 |
Genre | : Technology & Engineering |
ISBN | : 9814303208 |
While several reviews and books on surface nanophotonics and fluorescence spectroscopy are available, an updated focus on molecular plasmonics, including both theoretical methods and experimental aspects, is still lacking. This handbook is a comprehensive overview on the physics of the plasmon–emitter interaction, ranging from electromagnetism to quantum mechanics, from metal-enhanced fluorescence to surface-enhanced Raman scattering, from optical microscopy to synthesis of metal nanoparticles, filling the gap in the literature of this merging field. It allows experimentalists to have a solid theoretical reference at a different level of accuracy, and theoreticians to find new stimuli for novel computational methods and emerging applications.