Membrane proteins. Volume 557, Engineering, purification and crystallization

✍ Scribed by Shukla, Arun K

• Publisher: Academic Press
• Year: 2015
• Tongue: English
• Leaves: 620
• Series: Methods in Enzymology Volume 557
• Edition: 1
• Category: Library

✦ Synopsis

Membrane Proteins - Engineering, Purification and Crystallization, a volume of Methods In Enzymology, encompasses chapters from the leading experts in the area of membrane protein biology. The chapters provide a brief overview of the topics covered and also outline step-by-step protocol for the interested audience. Illustrations and case example images are included wherever appropriate to help the readers understand the schematics and general experimental outlines.

• Volume of Methods In Enzymology
• Contains a collection of a diverse array of topics in the area of membrane protein biology ranging from recombinant expression, isolation, functional characterization, biophysical studies and crystallization

✦ Table of Contents

Content: Front Cover
Membrane Proteins-Engineering, Purification and Crystallization
Copyright
Contents
Contributors
Preface
Section I: Membrane Protein Engineering, Solubilization and Purification
Chapter One: Multicolor Fluorescence-Based Screening Toward Structural Analysis of Multiprotein Membrane Complexes
1. Introduction
1.1. Screening approaches for membrane proteins and membrane protein complexes
1.2. Multicolor fluorescence-based screening of multiprotein membrane complexes 1.3. The major histocompatibility complex class I peptide-loading complex: A challenge for structural biology of membrane...2. Production of Fluorescently Labeled TAP Complexes in P. pastoris
2.1. Construct design
2.2. Transformation
2.3. Colony screening
2.4. Large-scale expression
2.5. Orthogonal purification
3. Production of Fluorescently Labeled TAP Complexes in Mammalian Cells
3.1. Construct design
3.2. Transfection
3.3. Orthogonal purification
4. Multicolor Fluorescence-Based Screening Approaches
4.1. MC-FSEC analysis
4.2. In-gel fluorescence and immunoblotting 4.3. Monitoring peptide binding to TAP4.4. Measurement of MHC I surface presentation by flow cytometry
5. Summary
Acknowledgments
References
Chapter Two: A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs
1. Introduction
2. Overall Strategy
3. Plasmids, Yeast Strains, and Media
3.1. Yeast strains
3.2. Plasmids
3.3. Media
4. Library Construction
4.1. Generation of T4L-containing cassettes with variable lengths of flanking regions
4.2. Insertion of cassettes containing the T4L gene into the replacement region of the receptor 2. MP Stability in Membrane Architecture3. Conventional Detergents
3.1. Detergent classification
3.2. Use of conventional detergents for MP study
3.3. Limitation of conventional detergents
4. Novel Amphipathic Systems
4.1. Detergent properties for MP structural study
4.2. Small amphipathic compounds
4.2.1. Conventional detergent variants
4.2.2. Hemifluorinated surfactants
4.2.3. Tripod amphiphiles
4.2.4. Facial amphiphiles
4.2.5. Rigid hydrophobic group-bearing amphiphiles
4.2.6. Neopentyl glycol (NG) class amphiphiles
4.3. Peptide-based amphiphiles