Protein Folding and Misfolding
Protein Folding and Misfolding

Author: Heinz Fabian

Publisher: Springer Science & Business Media

Published: 2011-09-18

Total Pages: 257

ISBN-13: 3642222307

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Infrared spectroscopy is a new and innovative technology to study protein folding/misfolding events in the broad arsenal of techniques conventionally used in this field. The progress in understanding protein folding and misfolding is primarily due to the development of biophysical methods which permit to probe conformational changes with high kinetic and structural resolution. The most commonly used approaches rely on rapid mixing methods to initiate the folding event via a sudden change in solvent conditions. Traditionally, techniques such as fluorescence, circular dichroism or visible absorption are applied to probe the process. In contrast to these techniques, infrared spectroscopy came into play only very recently, and the progress made in this field up to date which now permits to probe folding events over the time scale from picoseconds to minutes has not yet been discussed in a book. The aim of this book is to provide an overview of the developments as seen by some of the main contributors to the field. The chapters are not intended to give exhaustive reviews of the literature but, instead to illustrate examples demonstrating the sort of information, which infrared techniques can provide and how this information can be extracted from the experimental data. By discussing the strengths and limitations of the infrared approaches for the investigation of folding and misfolding mechanisms this book helps the reader to evaluate whether a particular system is appropriate for studies by infrared spectroscopy and which specific advantages the techniques offer to solve specific problems.

Fluorescence and Energy Transfer Studies of Membrane Protein Folding
Fluorescence and Energy Transfer Studies of Membrane Protein Folding

Author: Guipeun Kang

Publisher:

Published: 2015

Total Pages: 179

ISBN-13: 9781321852677

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Approximately 30 % of human genes encode for membrane proteins. Membrane proteins play important roles in cells as ion pumps, ligand receptors, and ion channels, and are targets for approximately 60 % of all therapeutic drugs. Despite their relevance in biology and biochemistry, membrane proteins account for only 1 % of the total number of reported protein structures (RCSB Protein Data Bank), and only 16 % of all protein folding literature is related to membrane protein folding (literature search from 1990 -- 2015, Web of Science). This dissertation presents spectroscopic studies of the in vitro folding mechanisms of outer membrane protein A (OmpA). Several optical tools are utilized, including circular dichroism (CD), tryptophan fluorescence, Förster resonance energy transfer (FRET), and ultraviolet resonance Raman (UVRR) spectroscopy. An improved method to determine free energies of unfolding of OmpA based on spectral decomposition is presented. Dynamics of OmpA folding in synthetic lipid bilayers of small unilamellar vesicles (SUVs) are investigated through studies of secondary and tertiary structures. CD and FRET data indicate that secondary and tertiary structures are formed within the first hour of folding, and strand extension and equilibration continues on a longer timescale. UVRR data complement the CD and FRET results, and reveal evolution of molecular interactions during folding. In particular, a tryptophan residue in the extra-vesicle portion of the pore (position 129) displays unusually intense Raman activity in the hydrogen-out-of-plane (HOOP) region. The increase in HOOP intensity is hypothesized to reflect perturbation of the indole ring electron density because of a nearby charged residue or hydroxyl group on neighboring threonine residue. More likely, hydrogen bonding of [pi] electrons on tryptophan with hydroxyl group contributes to the overall stability in addition to hydrophobic contacts by neighboring hydrophobic residues. A relatively new folding environment of nanodiscs is also explored. Preliminary FRET and UVRR data show that OmpA folds and inserts into nanodiscs. Collectively, these measurements elucidate changes in secondary and tertiary structures as well as molecular interactions of tryptophan residues during membrane protein folding.

Circular Dichroism and the Conformational Analysis of Biomolecules
Circular Dichroism and the Conformational Analysis of Biomolecules

Author: G.D. Fasman

Publisher: Springer Science & Business Media

Published: 2013-11-11

Total Pages: 739

ISBN-13: 1475725086

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''Excellent and very timely....It will undoubtedly become a standard reference for the application of circular dichroism (CD) to biomolecules.'' --- Quarterly Review of Biology, March 1997 ''[T]estament to the book's utility is the fact that during the course of my review I had to 'rescue' it from the desks of graduate students on an almost daily basis. In summary, this is a great book.'' --- American Scientist ''Well documented chapters provide a very good insight into the problems surrounding the conformation of biomacromolecules...An indispensible source of information.'' --- Nahrung, 42(2), 1998 Renowned experts present the first state-of-the-art description of circular dichroism spectroscopy (CD). Chapters present in-depth discussions of the history of the field, the theory of CD for application to globular proteins, membrane proteins, peptides, nucleic acids and their interactions, carbohydrates, and instrumentation. Discussions also feature new techniques using synchrotron radiation, vibrational Raman optical activity, and vibrational CD. More than 250 illustrations supplement the text.

Structural Genomics
Structural Genomics

Author: Yu Wai Chen

Publisher: Humana

Published: 2013-11-09

Total Pages: 0

ISBN-13: 9781627036900

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The field of Structural Genomics has produced many technological advances that transform and accelerate structure solution and analysis. Structural Genomics: General Applications emphasizes the benefits to the wider structural research community. It also reflects the current trend in tackling the more ambitious challenges of studying macromolecular machineries and complexes. Divided into three convenient sections, topics include the cloning and production of proteins for structural studies, experimental methods, and computational methods and data analysis. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Structural Genomics: General Applications aims primarily to channel spin-off technologies to the average structural biologist in a small or medium-sized laboratory.

Touring the Conformational Landscape of Proteins with High-Pressure SDSL EPR Spectroscopy
Touring the Conformational Landscape of Proteins with High-Pressure SDSL EPR Spectroscopy

Author: Michael Lerch

Publisher:

Published: 2015

Total Pages: 251

ISBN-13:

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Proteins in solution are dynamic molecules that exhibit conformational flexibility across a range of time and length scales, and characterizing the functional role of protein motion is a primary goal in molecular biophysics. High hydrostatic pressure has emerged as a powerful probe of protein conformational flexibility. Development of instrumentation and methodologies that enable electron paramagnetic resonance (EPR) experiments on proteins at high pressure is the central aim of the work presented in this dissertation. Pressurization of proteins reveals regions of elevated compressibility, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that "invisible" excited states become accessible for spectroscopic characterization. Current evidence indicates that pressure cleanly shifts the relative populations of states solely according to differences in partial molar volume without altering the shape of the conformational free energy landscape. Thus, variable pressure is a powerful tool for dissecting details of the landscape, and site-directed spin labeling coupled with electron paramagnetic resonance spectroscopy (SDSL EPR) is an ideal strategy in terms of sensitivity and time scale to detect the effects of pressure and interpret them in terms of structure and dynamics. In this dissertation, newly developed high-pressure instrumentation for both variable-pressure continuous-wave EPR and pressure-resolve double electron-electron resonance (PR DEER) of proteins in aqueous solution is described. The applications presented illustrate the considerable potential of the methods to: (1) identify compressible (flexible) regions in a folded protein; (2) determine thermodynamic parameters that relate conformational states in equilibrium; (3) populate and characterize excited states of proteins undetected at atmospheric pressure; (4) reveal the structural heterogeneity of conformational ensembles and provide distance constraints on the global structure of pressure-populated states. The SDSL EPR results are complemented by global secondary structure information provided by high-pressure circular dichroism experiments. This work lays the foundation for future developments in high-pressure SDSL EPR, including pressure-jump relaxation spectroscopy to determine the lifetime of conformational states in the millisecond range and high pressure saturation recovery exchange spectroscopy to enable measurement of lifetimes of states in the microsecond range. SDSL EPR has unique advantages for the study of membrane proteins in their native environment under physiological conditions, and applications of high-pressure SDSL EPR to explore the conformational equilibria and dynamics of integral membrane proteins is a high priority for future work.