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.

Protein Folding Handbook, 5 Volume Set
Protein Folding Handbook, 5 Volume Set

Author: Johannes Buchner

Publisher: Wiley-VCH

Published: 2005-03-11

Total Pages: 2623

ISBN-13: 9783527307845

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How a polypeptide chain folds into a stable and functional protein is probably the most important question in present-day molecular biology. Reliably predicting the folding process allows to deduce protein function from genomic information alone and will bring about a revolution in structural genomics. Understanding the way in which proper protein folding is controlled by the cell is required to find a cure for Alzheimer's and other diseases caused by misfolded proteins. This unique handbook contains the expertise from more than 60 research groups, covering the entire range of topics in protein folding - from biophysics to molecular medicine. The first part explains the principles and factors governing protein stability, and how this knowledge may be used to predict folding pathways. It also surveys important techniques used to study the protein folding process, including spectroscopic, chemical and biological techniques. The second part is devoted to protein folding, unfolding, and misfolding in the cellular context, introducing chaperones and other enzymes involved in protein folding, as well as a study of the pathophysiology of misfolded proteins in amyloid and other disease states. The whole is rounded off by a discussion of the possibility of interfering with the protein folding process by genetic engineering. The comprehensiveness and outstanding quality of the carefully selected contents make this the ultimate reference for every scientist with an interest in protein folding.

Single-Molecule Fluorescence Spectroscopy of the Folding of a Repeat Protein
Single-Molecule Fluorescence Spectroscopy of the Folding of a Repeat Protein

Author: Sharona Cohen

Publisher: Springer

Published: 2015-10-17

Total Pages: 70

ISBN-13: 3319095587

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In this thesis single-molecule fluorescence resonance energy transfer (FRET) spectroscopy was used to study the folding of a protein that belongs to the large and important family of repeat proteins. Cohen shows that the dynamics of the expanded conformations is likely to be very fast, suggesting a spring-like motion of the whole chain. The findings shed new light on the elasticity of structure in repeat proteins, which is related to their function in binding multiple and disparate partners. This concise research summary provides useful insights for students beginning a PhD in this or a related area, and researchers entering this field.

Fluorescence Studies on Biological Membranes
Fluorescence Studies on Biological Membranes

Author: Herwig J. Hilderson

Publisher: Springer Science & Business Media

Published: 2012-12-06

Total Pages: 480

ISBN-13: 1461393590

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As stated by its first editor, Dr. D. B. Roodyn, the primary goal of the series Subcellular Biochemistry is to achieve an integrated view of the cell by bringing together results from a wide range of different techniques and disciplines. This volume deals with the applications of fluorescence spectroscopy to membrane research. It seeks to present complementary biochemical and bio physical data on both the structure and the dynamics of biological membranes. Biophysics and biochemistry are improving more and more in their ability to study biomembranes, overlapping somewhat in this area and explaining the functioning of the whole cell in terms of the properties of its individual com ponents. Therefore, we have brought together an international group of experts in order to report on and review advances in fluorescence studies on biological membranes, thereby highlighting subcellular aspects. The first chapters present a critical evaluation of the current applications of dynamic and steady-state fluorescence techniques. Subsequent chapters dis cuss more specific applications in cells, biological membranes, and their con stituents (lipids, proteins).

Fluorescent Methods to Study Biological Membranes
Fluorescent Methods to Study Biological Membranes

Author: Yves Mely

Publisher: Springer Science & Business Media

Published: 2012-10-10

Total Pages: 484

ISBN-13: 3642331289

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Biological membranes play a central role in cell structure, shape and functions. However, investigating the membrane bilayer has proved to be difficult due to its highly dynamic and anisotropic structure, which generates steep gradients at the nanometer scale. Due to the decisive impact of recently developed fluorescence-based techniques, tremendous advances have been made in the last few years in our understanding of membrane characteristics and functions. In this context, the present book illustrates some of these major advances by collecting review articles written by highly respected experts. The book is organized in three parts, the first of which deals with membrane probes and model membranes. The second part describes the use of advanced quantitative and high-resolution techniques to explore the properties of biological membranes, illustrating the key progress made regarding membrane organization, dynamics and interactions. The third part is focused on the investigation of membrane proteins using the same techniques, and notably on the membrane receptors that play a central role in signaling pathways and therapeutic strategies. All chapters provide comprehensive information on membranes and their exploration for beginners in the field and advanced researchers alike.

Fluorescent Proteins
Fluorescent Proteins

Author: Kevin F. Sullivan

Publisher: Elsevier

Published: 2007-12-14

Total Pages: 613

ISBN-13: 0080557244

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This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.

Single Particle Tracking and Single Molecule Energy Transfer
Single Particle Tracking and Single Molecule Energy Transfer

Author: Christoph Bräuchle

Publisher: John Wiley & Sons

Published: 2009-10-30

Total Pages: 359

ISBN-13: 9783527628377

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Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.

Protein Folding Protocols
Protein Folding Protocols

Author: Yawen Bai

Publisher: Springer Science & Business Media

Published: 2008-02-04

Total Pages: 332

ISBN-13: 1597451894

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Covering experiment and theory, bioinformatics approaches, and state-of-the-art simulation protocols for better sampling of the conformational space, this volume describes a broad range of techniques to study, predict, and analyze the protein folding process. Protein Folding Protocols also provides sample approaches toward the prediction of protein structure starting from the amino acid sequence, in the absence of overall homologous sequences.