Vibrational Spectroscopy In Protein Research

Protein research is a frontier field in science. Proteins are widely distributed in plants and animals and are the principal constituents of the protoplasm of all cells, and consist essentially of combinations of a-amino acids in peptide linkages. Twenty different amino acids are commonly found in proteins, and serve as enzymes, structural elements, hormones, immunoglobulins, etc., and are involved throughout the body, and in photosynthesis. This book gathers new leading-edge research from throughout the world in this exciting and exploding field of research.

Vibrational Spectroscopy in Protein Research offers a thorough discussion of vibrational spectroscopy in protein research, providing researchers with clear, practical guidance on methods employed, areas of application, and modes of analysis. With chapter contributions from international leaders in the field, the book addresses basic principles of vibrational spectroscopy in protein research, instrumentation and technologies available, sampling methods, quantitative analysis, origin of group frequencies, and qualitative interpretation. In addition to discussing vibrational spectroscopy for the analysis of purified proteins, chapter authors also examine its use in studying complex protein systems, including protein aggregates, fibrous proteins, membrane proteins and protein assemblies. Emphasis throughout the book is placed on applications in human tissue, cell development, and disease analysis, with chapters dedicated to studies of molecular changes that occur during disease progression, as well as identifying changes in tissues and cells in disease studies. Provides thorough guidance in implementing cutting-edge vibrational spectroscopic methods from international leaders in the field Emphasizes in vivo, in situ and non-invasive analysis of proteins in biomedical and life science research more broadly Contains chapters that address vibrational spectroscopy for the study of simple purified proteins and protein aggregates, fibrous proteins, membrane proteins and protein assemblies

The authors describe basic theoretical concepts of vibrational spectroscopy, address instrumental aspects and experimental procedures, and discuss experimental and theoretical methods for interpreting vibrational spectra. It is shown how vibrational spectroscopy provides information on general aspects of proteins, such as structure, dynamics, and protein folding. In addition, the authors use selected examples to demonstrate the application of Raman and IR spectroscopy to specific biological systems, such as metalloproteins, and photoreceptors. Throughout, references to extensive mathematical and physical aspects, involved biochemical features, and aspects of molecular biology are set in boxes for easier reading. Ideal for undergraduate as well as graduate students of biology, biochemistry, chemistry, and physics looking for a compact introduction to this field.

Theoretical Vibrational Spectroscopy of Proteins Lu Wang Under the supervision of Professor James L. Skinner At the University of Wisconsin-Madison Vibrational spectroscopy, such as linear and two-dimensional infrared (IR) spectroscopy, is widely utilized to study the structure and dynamics of peptides and proteins. Interpretation of the experiment, or a direct assignment of the complex experimental spectra to the underlying protein structure, can be difficult. Molecular dynamics (MD) simulations offer a complementary approach to provide high-resolution structural and temporal information of proteins, although they are limited by factors such as force field accuracy and are not directly comparable to spectroscopic experiments. We have developed vibrational frequency maps for proteins that generate instantaneous site frequencies directly from MD simulations. We combine the frequency maps with established nearest-neighbor frequency shift and coupling schemes and a mixed quantum/classical framework to form a theoretical strategy for calculating protein linear and 2D IR spectra in the amide I region. This theoretical method provides a means to bridge spectroscopic experiments and molecular simulations, which allows a critical assessment of MD simulations by comparison to experiment, and enables the interpretation of experimental spectra at the molecular level. In this dissertation, we present the development of the vibrational frequency maps and provide the theoretical protocol that allows the calculation of protein vibrational spectra directly from MD simulations. We validate the theoretical method by applying it to peptides with various secondary structures in aqueous solution, and apply it to a few biologically relevant problems. For instance, we have studied the thermal unfolding transition of the villin headpiece subdomain (HP36) using IR spectra calculations. We follow the unfolding process of HP36 by monitoring its spectral changes as a function of temperature. With the help of isotope labeling, we are able to capture the feature that helix 2 of HP36 loses its secondary structure before global unfolding occurs, in agreement with experiment. In collaboration with the Zanni group and the de Pablo group at University of Wisconsin, we have also carried out studies on IAPP, a peptide closely related to type 2 diabetes. By combining theoretical modeling with extensive computer simulations and spectroscopic experiments, we have investigated the structure and dynamics of IAPP in aqueous solution, in the fibril form and in the vicinity of lipid membranes.

In recent years there has been a tremendous growth in the use of vibrational spectroscopic methods for diagnosis and screening. These applications range from diagnosis of disease states in humans, such as cancer, to rapid identification and screening of microorganisms. The growth in such types of studies has been possible thanks to advances in instrumentation and associated computational and mathematical tools for data processing and analysis. This volume of Advances in Biomedical Spectroscopy contains chapters from leading experts who discuss the latest advances in the application of Fourier transform infrared (FTIR), Near infrared (NIR), Terahertz and Raman spectroscopy for diagnosis and screening in fields ranging from medicine, dentistry, forensics and aquatic science. Many of the chapters provide information on sample preparation, data acquisition and data interpretation that would be particularly valuable for new users of these techniques including established scientists and graduate students in both academia and industry.

Bringing several disparate aspects of food science and analysis together in one place, Applications of Vibrational Spectroscopy to Food Science provides a comprehensive, state-of the-art text presenting the fundamentals of the methodology, as well as underlying current areas of research in food science analysis. All of the major spectroscopic techniques are also covered – showing how each one can be used beneficially and in a complementary approach for certain applications. Case studies illustrate the many applications in vibrational spectroscopy to the analysis of foodstuffs.

Simulations of two-dimensional infrared (2DIR) spectroscopy of several proteins are presented. Applications of 2DIR spectroscopy to protein folding, protein aggregation, and photosensing are reported. We demonstrate that 2DIR spectroscopy is an excellent probe of protein structure and dynamics. Our simulations predict future experiments as well as provide detailed explanations of previous experiments. We also present simulations of the related two-dimensional ultraviolet and two-dimensional stimulated resonance Raman spectroscopies, which are shown to provide complementary information to 2DIR spectroscopy. This thesis can be viewed as a guide for the design and analysis of future two-dimensional optical measurements on proteins.

In recent years there has been a tremendous growth in the use of vibrational spectroscopic methods for diagnosis and screening. These applications range from diagnosis of disease states in humans, such as cancer, to rapid identification and screening of microorganisms. The growth in such types of studies has been possible thanks to advances in instrumentation and associated computational and mathematical tools for data processing and analysis. This volume of Advances in Biomedical Spectroscopy contains chapters from leading experts who discuss the latest advances in the application of Fourier transform infrared (FTIR), Near infrared (NIR), Terahertz and Raman spectroscopy for diagnosis and screening in fields ranging from medicine, dentistry, forensics and aquatic science. Many of the chapters provide information on sample preparation, data acquisition and data interpretation that would be particularly valuable for new users of these techniques including established scientists and graduate students in both academia and industry.

Modern Vibrational Spectroscopy and Micro-Spectroscopy: Theory, Instrumentation and Biomedical Applications unites the theory and background of conventional vibrational spectroscopy with the principles of microspectroscopy. It starts with basic theory as it applies to small molecules and then expands it to include the large biomolecules which are the main topic of the book with an emphasis on practical experiments, results analysis and medical and diagnostic applications. This book is unique in that it addresses both the parent spectroscopy and the microspectroscopic aspects in one volume. Part I covers the basic theory, principles and instrumentation of classical vibrational, infrared and Raman spectroscopy. It is aimed at researchers with a background in chemistry and physics, and is presented at the level suitable for first year graduate students. The latter half of Part I is devoted to more novel subjects in vibrational spectroscopy, such as resonance and non-linear Raman effects, vibrational optical activity, time resolved spectroscopy and computational methods. Thus, Part 1 represents a short course into modern vibrational spectroscopy. Part II is devoted in its entirety to applications of vibrational spectroscopic techniques to biophysical and bio-structural research, and the more recent extension of vibrational spectroscopy to microscopic data acquisition. Vibrational microscopy (or microspectroscopy) has opened entirely new avenues toward applications in the biomedical sciences, and has created new research fields collectively referred to as Spectral Cytopathology (SCP) and Spectral Histopathology (SHP). In order to fully exploit the information contained in the micro-spectral datasets, methods of multivariate analysis need to be employed. These methods, along with representative results of both SCP and SHP are presented and discussed in detail in Part II.

This dissertation, "Study of Chemically Modified Food Proteins by Vibrational Spectroscopy" by Hing-wan, Wong, 王慶雲, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled STUDY OF CHEMICALLY MODIFIED FOOD PROTEINS BY VIBRATIONAL SPECTROSCOPY Submitted by Wong Hing Wan for the degree of Doctor of Philosophy at The University of Hong Kong in August 2006 The Raman and Fourier-transform infrared (FTIR) vibrational spectroscopic methods were used to study chemically modified food proteins. Four chemical modification methods: acid deamidation, tryptophan-amidation, sulfitolysis, and trypsin-hydrolysis, and several widely-used food protein products: soy protein isolates (SPI), spray-dried egg white powders (EW), whey protein isolates (WPI), gluten, and casein were selected for study. Raman and FTIR spectra of the chemically modified proteins showed characteristic -1 marker bands. A new Raman C=O stretch vibration band at 1780 cm was observed in deamidated proteins, and was attributed to the γ-carboxyl groups of aspartic and -1 glutamic acids. Similarly, a phenyl stretch vibration at 1552 cm (Raman) was found in - -1 amidated proteins, a -S-SO (thiosulfate) stretch vibration at 1028 cm (in both Raman and FTIR) was found in disulfide bond cleaved samples, and a C=O stretch vibration at -1 -1 1732 cm (Raman) and 1746 cm (FTIR) was observed in trypsin-hydrolyzed proteins. The intensity of these marker bands was found to increase with increases in the level of chemical modification. Calibration curves were constructed by plotting the ratio of the -1 intensity of a particular marker band to the intensity of a Raman 1003 cm -1 phenylalanine stretching band or a FTIR 2116 cm ferricyanide stretching band (used as internal standards) against the extent of modification determined by conventional wet chemistry methods. Linear fits were obtained with correlation coefficients (r) >0.98 and > 0.94 for the Raman and FTIR calibration curves, respectively, indicating strong linear relationships between the marker band intensities and the levels of modification for all the modified protein products. Advantages of the newly developed Raman and FTIR methods over wet chemistry methods are simple and rapid sample preparation, fast determination, and utilization of relatively safe chemicals. Hence, Raman and FTIR spectroscopy have the potential to be further developed for quality control in the food processing industry. The effects of chemical modifications on the conformation and molecular structure of food proteins were studied by vibrational spectroscopy, supplemented by circular dichroism spectroscopy and laser light scattering. Deamidation increased the negative charges in the proteins, resulting in pronounced conformational changes including exposure of hydrophobic residues, increases in disordered conformations and formation of aggregated molecules with compact structures. Amidation also led to increases in disordered structures, possibly due to the attachment of bulky non-polar tryptophan residues. Sulfitolysis breaks up disulfide bonds in proteins, leading to increases in random coil structures and disaggregation of molecules. Hydrolyzed proteins showed marked spectral changes in the amide I and C-H bending vibrations, and progressive increases in random coil structures with concomitant decreases in ordered secondary structure components, suggesting protein denaturation due to cleavage of the peptide bonds. The present study demonstrates the wide application of Raman and FTIR