Membrane Transport Piece by Piece: Production of Transmembrane Peptides for Structural and Functional Studies

Grant Kemp1, Larry Fliegel1, Howard S. Young2

1 Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, 2 National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.8
DOI:  10.1002/0471140864.ps2908s75
Online Posting Date:  February, 2014
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Abstract

Membrane proteins are involved in all cellular processes from signaling cascades to nutrient uptake and waste disposal. Because of these essential functions, many membrane proteins are recognized as important, yet elusive, clinical targets. Recent advances in structural biology have answered many questions about how membrane proteins function, yet one of the major bottlenecks remains the ability to obtain sufficient quantities of pure and homogeneous protein. This is particularly true for human membrane proteins, where novel expression strategies and structural techniques are needed to better characterize their function and therapeutic potential. One way to approach this challenge is to determine the structure of smaller pieces of membrane proteins that can be assembled into models of the complete protein. This unit describes the rationale for working with single or multiple transmembrane segments and provides a description of strategies and methods to express and purify them for structural and functional studies using a maltose binding protein (MBP) fusion. The bulk of the unit outlines a detailed methodology and justification for producing these peptides under native‐like conditions. Curr. Protoc. Protein Sci. 75:29.8.1‐29.8.28 © 2014 by John Wiley & Sons, Inc.

Keywords: membrane proteins; hydrophobic peptides; bacterial expression; maltose binding protein; organic extraction; molecular structure

     
 
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Table of Contents

  • Introduction
  • Strategic Planning: Designing a Peptide for Expression
  • Basic Protocol 1: Vector Construction and Cloning of a Transmembrane Peptide for Expression
  • Basic Protocol 2: Expression and Purification of Fusion Protein
  • Basic Protocol 3: Recovery and Further Purification of the Cleaved Hydrophobic Peptide
  • Alternate Protocol 1: High‐Performance Liquid Chromatography Purification of the Hydrophobic Peptide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Vector Construction and Cloning of a Transmembrane Peptide for Expression

  Materials
  • 10 μM specific PCR forward primer (Integrated DNA Technologies (http://www.idtdna.com; see note below for suggestions on primer design)
  • T4 kinase kit (New England Biolabs, cat. no. M0201S)
  • 10 μM specific PCR reverse primer (Integrated DNA Technologies, http://www.idtdna.com)
  • cDNA template containing the gene of interest
  • DNA polymerase (high fidelity, such as Pfx, preferred; Invitrogen, cat. no. 11708‐013) and its specific buffers (also see )
  • 10 mM dNTP mix (Fermentas, cat. no. R0181, or see recipe in )
  • QIAquick PCR Purification Kit (Qiagen, cat. no. 28104)
  • QIAquick Gel Extraction Kit (Qiagen, cat. no. 28704)
  • Restriction enzymes: PdmI (Thermo Scientific, cat. no. FD1534), EcoRI (Thermo Scientific, cat. no. FD0274), BamHI (Thermo Scientific, cat. no. cat. no. FD0054), and respective buffers
  • pMal‐c5X plasmid (New England Biolabs, cat. no. N8108)
  • T4 DNA ligation kit (New England Biolabs, cat. no. M0202S)
  • Competent DH5α E. coli (Invitrogen, cat. no. 18258‐012; stored at −80°C)
  • LB+amp plates (see recipe)
  • 80% (v/v) glycerol (autoclaved)
  • Qiagen Plasmid MidiPrep Kit (Qiagen, cat. no. 12143)
  • pMal sequencing primers (New England Biolabs)
  • 200‐μl PCR tubes
  • Thermal cycler
  • Sterile loop
  • 100°C heat block
  • 37°C shaking incubator
  • 1.5‐ml cryotubes (Nunc, cat. no. 114858) for glycerol stocks
  • Additional reagents and equipment for PCR ( ), agarose gel electrophoresis ( ), and DNA sequencing (Shendure et al., )
NOTE: At any stage, purified DNA (without added enzymes) can be stored at 4°C for a few days or at −20°C for several months creating convenient stopping points.

Basic Protocol 2: Expression and Purification of Fusion Protein

  Materials
  • Plate containing positive transformants (from protocol 1, step 11, or fresh LB+amp plate streaked using glycerol stocks from protocol 1, step 16)
  • LB+amp medium: add 1 ml of 100 mg/ml ampicillin stock (see recipe) per liter LB medium (see recipe)
  • 0.5 M isopropyl β‐D‐1‐thiogalactopyranoside (IPTG; filter sterilized, see recipe)
  • Lysis buffer (see recipe), cold
  • Amylose resin (maltose affinity resin; New England Biolabs, cat. no. E8021L)
  • Purification buffer (see recipe)
  • 1× Bradford reagent (BioRad, cat. no. 500‐0205)
  • Elution buffer (see recipe)
  • 0.1% (w/v) sodium dodecyl sulfate (SDS)
  • 0.02% sodium azide (NaN 3) or 20% (v/v) ethanol for column storage
  • Tobacco Etch Virus (TEV) protease (Sigma‐Aldrich, cat. no. T4455)
  • 1 M dithiothreitol (DTT; see recipe)
  • 10‐ml sterile culture tubes with caps (Simport, cat. no. T406‐2A, http://www.simport.com/)
  • Sterile loop
  • 37°C shaking incubator
  • 50‐ml Erlenmeyer flasks with caps or foil coverings, sterile
  • Spectrophotometer for reading optical density (600 nm)
  • Refrigerated centrifuge and ultracentrifuge with appropriate bottles/tubes
  • Cell lysis apparatus: e.g., sonicator, French press, high‐pressure homogenizer (Emulsiflex); see Commentary for more information
  • Nutator or other apparatus for batch incubation of lysate with amylose resin
  • Gravity purification column and caps (49‐ml glass Econo‐Columns, BioRad, cat. no. 737‐2512)
  • Tubes for collecting column flowthrough, washes, and eluates
  • Filter‐driven concentrator apparatus (Amicon Stirred Cell, Millipore, cat. no. 5124) and ultrafiltration membranes (MWCO 10,000; Millipore, cat. no. PLGC07610)
  • 16°C incubator for TEV cleavage
  • Additional reagents and equipment for SDS‐PAGE (unit )

Basic Protocol 3: Recovery and Further Purification of the Cleaved Hydrophobic Peptide

  Materials
  • Protease digestion reaction (output of protocol 2)
  • 60% trichloroacetic acid (TCA)
  • Chloroform
  • Isopropanol
  • Centrifuge, rotor, and glass tubes capable of centrifugation at 9000 × g
  • Glass rod
  • Metal spatula to resuspend precipitated protein pellet
  • Glass Dounce homogenizer (Fisher Scientific, cat. no. FB56699)
  • 125‐ml separatory funnel
  • Teflon‐lined screw‐cap glass tubes for collecting organic extract
  • Rotary evaporator
  • Nitrogen gas tank
  • Heat block and/or lyophilizer
  • Additional reagents and equipment for Tris‐Tricine SDS‐PAGE (unit )
IMPORTANT NOTE: The following steps should be done using ONLY chloroform‐insensitive materials like glass, metal, and Teflon. Many plastics dissolve in chloroform and will contaminate the sample. To simplify the following steps, all the volumes given are for 100 mg of fusion protein. Increase the volumes accordingly for different starting amounts.

Alternate Protocol 1: High‐Performance Liquid Chromatography Purification of the Hydrophobic Peptide

  Materials
  • Protein pellet ( protocol 3, step 4)
  • 1 M and 7 M buffered GuHCl (see recipe)
  • Reversed‐phase solvents:
    • Solvent A (H 2O‐TFA; see recipe)
    • Solvent B (isopropanol‐TFA; see recipe)
  • Dounce homogenizer (Fisher Scientific, cat. no. FB56699)
  • Refrigerated centrifuge, rotor and bottles capable of 10,000 × g at 4°C
  • Reversed‐phase semi‐preparative HPLC column (Zorbax SB300 C8 semi‐preparative column, Agilent Technologies, cat. no. SB300 C8)
  • HPLC equipped with column heater and fraction collector
  • Additional reagents and equipment for Tris‐Tricine SDS‐PAGE (unit ) and reversed‐phase separation of peptides (unit )
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Figures

Videos

Literature Cited

Literature Cited
   Afara, M.R. , Trieber, C.A. , Glaves, J.P. , and Young, H.S. 2006. Rational design of peptide inhibitors of the sarcoplasmic reticulum calcium pump. Biochemistry 45:8617‐8627.
   Afara, M.R. , Trieber, C.A. , Ceholski, D.K. , and Young, H.S. 2008. Peptide inhibitors use two related mechanisms to alter the apparent calcium affinity of the sarcoplasmic reticulum calcium pump. Biochemistry 47:9522‐9530.
   Akabas, M.H. , Stauffer, D.A. , Xu, M. , and Karlin, A. 1992. Acetylcholine receptor channel structure probed in cysteine‐substitution mutants. Science 258:307‐310.
   Albert, A.D. and Yeagle, P.L. 2002. Structural studies on rhodopsin. Biochim. Biophys. Acta 1565:183‐195.
   Bertani, G. 2004. Lysogeny at mid‐twentieth century: P1, P2, and other experimental systems. J. Bacteriol. 186:595‐600.
   Bhave, G. , Nadin, B.M. , Brasier, D.J. , Glauner, K.S. , Shah, R.D. , Heinemann, S.F. , Karim, F. , and Gereau, R.W. 2003. Membrane topology of a metabotropic glutamate receptor. J. Biol. Chem. 278:30294‐30301.
   Bichet, P. , Mollat, P. , Capdevila, C. , and Sarubbi, E. 2000. Endogenous glutathione‐binding proteins of insect cell lines: Characterization and removal from glutathione S‐transferase (GST) fusion proteins. Protein Expr. Purif. 19:197‐201.
   Bordag, N. and Keller, S. 2010. α‐Helical transmembrane peptides: A “Divide and Conquer” approach to membrane proteins. Chem. Phys. Lipids 163:1‐26.
   Ceholski, D.K. , Trieber, C.A. , Holmes, C.F.B. , and Young, H.S. 2012. Lethal, hereditary mutants of phospholamban elude phosphorylation by protein kinase a. J. Biol. Chem. 287:26596‐26605.
   Chopra, A. , Yeagle, P.L. , Alderfer, J.A. , and Albert, A.D. 2000. Solution structure of the sixth transmembrane helix of the G‐protein‐coupled receptor, rhodopsin. Biochim. Biophys. Acta 1463:1‐5.
   Cole, C. , Barber, J.D. , and Barton, G.J. 2008. The Jpred 3 secondary structure prediction server. Nucl. Acids Res. 36:W197‐W201.
   Cregg, J.M. , Tolstorukov, I. , Kusari, A. , Sunga, J. , Madden, K. , and Chappell, T. 2009. Expression in the yeast Pichia pastoris . Methods Enzymol. 463:169‐189.
   Cuff, J.A. and Barton, G.J. 1999. Evaluation and improvement of multiple sequence methods for protein secondary structure prediction. Proteins Struct. Funct. Bioinformat. 34:508‐519.
   Cunningham, F. and Deber, C.M. 2007. Optimizing synthesis and expression of transmembrane peptides and proteins. Methods 41:370‐380.
   Cybulski, L.E. and De Mendoza, D. 2011. Bilayer hydrophobic thickness and integral membrane protein function. Curr. Protein Peptide Sci. 12:760‐766.
   Daly, R. and Hearn, M.T. W. 2005. Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production. J. Mol. Recogn. 18:119‐138.
   De Bernardez Clark, E. 1998. Refolding of recombinant proteins. Curr. Opin. Biotechnol. 9:157.
   De Planque, M.R.R. and Killian, J.A. 2003. Protein‐lipid interactions studied with designed transmembrane peptides: Role of hydrophobic matching and interfacial anchoring. Mol. Membrane Biol. 20:271‐284.
   Ding, J. , Rainey, J.K. , Xu, C. , Sykes, B.D. , and Fliegel, L. 2006. Structural and functional characterization of transmembrane segment VII of the Na+/H+ exchanger isoform 1. J. Biol. Chem. 281:29817‐29829.
   Douglas, J.L. , Trieber, C.A. , Afara, M. , and Young, H.S. 2005. Rapid, high‐yield expression and purification of Ca2+‐ATPase regulatory proteins for high‐resolution structural studies. Protein Expr. Purif. 40:118‐125.
   Duff, K.C. and Ashley, R.H. 1992. The transmembrane domain of influenza A M2 protein forms amantadine‐sensitive proton channels in planar lipid bilayers. Virology 190:485‐489.
   Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557‐580.
   Hu, J. , Qin, H. , Li, C. , Sharma, M. , Cross, T.A. , and Gao, F.P. 2007. Structural biology of transmembrane domains: Efficient production and characterization of transmembrane peptides by NMR. Protein Sci. 16:2153‐2165.
   Hunt, J.F. , Earnest, T.N. , Bousché, O. , Kalghatgi, K. , Reilly, K. , Horváth, C. , Rothschild, K.J. , and Engelman, D.M. 1997. A biophysical study of integral membrane protein folding. Biochemistry 36:15156‐15176.
   Jidenko, M. , Nielsen, R.C. , Sørensen, T.L.‐M. , Møller, J.V. , le Maire, M. , Nissen, P. , and Jaxel, C. 2005. Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae . Proc. Natl. Acad. Sci. U.S.A. 102:11687‐11691.
   Jones, P.G. and Inouye, M. 1994. The cold‐shock response—a hot topic. Mol. Microbiol. 11:811‐818.
   Junge, F. , Schneider, B. , Reckel, S. , Schwarz, D. , Dötsch, V. , and Bernhard, F. 2008. Large‐scale production of functional membrane proteins. Cell. Mol. Life Sci. 65:1729‐1755.
   Kane, J.F. and Hartley, D.L. 1988. Formation of recombinant protein inclusion bodies in Escherichia coli . Trends Biotechnol. 6:95‐101.
   Kapust, R.B. and Waugh, D.S. 1999. Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8:1668‐1674.
   Karmazyn, M. , Avkiran, M. , and Fliegel, L. (eds.). 2003. The Sodium‐Hydrogen Exchanger: From Molecule to its Role in Disease. Kluwer Academic Publishers, Dordrecht, The Netherlands.
   Katragadda, M. , Alderfer, J.L. , and Yeagle, P.L. 2000. Solution structure of the loops of bacteriorhodopsin closely resembles the crystal structure. Biochim. Biophys. Acta 1466:1‐6.
   Katragadda, M. , Alderfer, J.L. , and Yeagle, P.L. 2001. Assembly of a polytopic membrane protein structure from the solution structures of overlapping peptide fragments of bacteriorhodopsin. Biophys. J. 81:1029‐1036.
   Klammt, C. , Schwarz, D. , Dötsch, V. , and Bernhard, F. 2007. Cell‐free production of integral membrane proteins on a preparative scale. In In Vitro Transcription and Translation Protocols Methods in Molecular Biology. ( G. Grandi , ed.) pp. 57‐78. Humana Press, Totowa, N.J. Available at: http://www.springerlink.com/content/l3262163j2827804/abstract/ [Accessed October 2, 2012].
   Kocherla, H. , Marino, J. , Shao, X. , Graf, J. , Zou, C. , and Zerbe, O. 2012. Biosynthesis and spectroscopic characterization of 2‐TM fragments encompassing the sequence of a human GPCR, the Y4 receptor. Chembiochem 13:818‐828.
   Krogh, A. , Larsson, B. , Von Heijne, G. , and Sonnhammer, E.L. 2001. Predicting transmembrane protein topology with a hidden markov model: Application to complete genomes. J. Mol. Biol. 305:567‐580.
   Landau, M. , Herz, K. , Padan, E. , and Ben‐Tal, N. 2007. Model structure of the Na+/H+ exchanger 1 (NHE1): Functional and clinical implications. J. Biol. Chem. 282:37854‐37863.
   Lee, B.L. , Li, X. , Liu, Y. , Sykes, B.D. , and Fliegel, L. 2009a. Structural and functional analysis of extracellular loop 2 of the Na(+)/H(+) exchanger. Biochim. Biophys. Acta 1788:2481‐2488.
   Lee, B.L. , Li, X. , Liu, Y. , Sykes, B.D. , and Fliegel, L. 2009b. Structural and functional analysis of transmembrane XI of the NHE1 isoform of the Na+/H+ exchanger. J. Biol. Chem. 284:11546‐11556.
   Lee, B.L. , Sykes, B.D. , and Fliegel, L. 2011. Structural analysis of the Na+/H+ exchanger isoform 1 (NHE1) using the divide and conquer approach. Biochem. Cell Biol. 89:189‐199.
   Liu, L.P. and Deber, C.M. 1998. Guidelines for membrane protein engineering derived from de novo designed model peptides. Biopolymers 47:41‐62.
   Lundstrom, K. 2010. Expression of mammalian membrane proteins in mammalian cells using Semliki Forest Virus vectors. In Heterologous Expression of Membrane Proteins Methods in Molecular Biology. ( I. Mus‐Veteau , ed.) pp. 149‐163. Humana Press, Totowa, N.J. Available at: http://www.springerlink.com/content/x18r02152686653r/abstract/ [Accessed October 2, 2012].
   Marblestone, J.G. , Edavettal, S.C. , Lim, Y. , Lim, P. , Zuo, X. , and Butt, T.R. 2006. Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Sci. 15:182‐189.
   Melnyk, R.A. , Partridge, A.W. , Yip, J. , Wu, Y. , Goto, N.K. , and Deber, C.M. 2003. Polar residue tagging of transmembrane peptides. Peptide Sci. 71:675‐685.
   Miroux, B. and Walker, J.E. 1996. Over‐production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260:289‐298.
   Mishima, M. , Wakabayashi, S. , and Kojima, C. 2007. Solution structure of the cytoplasmic region of Na+/H +exchanger 1 complexed with essential cofactor calcineurin B homologous protein 1. J. Biol. Chem. 282:2741‐2751.
   Moncoq, K. , Kemp, G. , Li, X. , Fliegel, L. , and Young, H.S. 2008. Dimeric structure of human Na+/H+ exchanger isoform 1 overproduced in Saccharomyces cerevisiae . J. Biol. Chem. 283:4145‐4154.
   Nagai, K. , Perutz, M.F. , and Poyart, C. 1985. Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli . Proc. Natl. Acad. Sci. U.S.A. 82:7252‐7255.
   Nallamsetty, S. and Waugh, D.S. 2006. Solubility‐enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr. Purif. 45:175‐182.
   Newby, Z.E.R. , O'Connell, J.D. , Gruswitz, F. , Hays, F.A. , Harries, W.E.C. , Harwood, I.M. , Ho, J.D. , Lee, J.K. , Savage, D.F. , Miercke, L.J.W. , and Stroud, R.M. 2009. A general protocol for the crystallization of membrane proteins for X‐ray structural investigation. Nat. Protoc. 4:619‐637.
   Nygaard, E.B. , Lagerstedt, J.O. , Bjerre, G. , Shi, B. , Budamagunta, M. , Poulsen, K.A. , Meinild, S. , Rigor, R.R. , Voss, J.C. , Cala, P.M. , and Pedersen, S.F. 2011. Structural modeling and electron paramagnetic resonance spectroscopy of the human Na+/H+ exchanger isoform 1, NHE1. J. Biol. Chem. 286:634‐648.
   Palczewski, K. , Kumasaka, T. , Hori, T. , Behnke, C.A. , Motoshima, H. , Fox, B.A. , Le Trong, I. , Teller, D.C. , Okada, T. , Stenkamp, R.E. , Yamamoto, M , and Miyano, M. 2000. Crystal structure of rhodopsin: A G protein‐coupled receptor. Science 289:739‐745.
   Punta, M. , Forrest, L.R. , Bigelow, H. , Kernytsky, A. , Liu, J. , and Rost, B. 2007. Membrane protein prediction methods. Methods 41:460‐474.
   Raman, P. , Cherezov, V. , and Caffrey, M. 2006. The membrane protein data bank. Cell. Mol. Life Sci. 63:36‐51.
   Reddy, G.L. , Iwamoto, T. , Tomich, J.M. , and Montal, M. 1993. Synthetic peptides and four‐helix bundle proteins as model systems for the pore‐forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore‐lining structure. J. Biol. Chem. 268:14608‐14615.
   Reddy, L.G. , Jones, L.R. , Cala, S.E. , O'Brian, J.J. , Tatulian, S.A. , and Stokes, D.L. 1995. Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+‐ATPase. J. Biol. Chem. 270:9390‐9397.
   Reddy, T. , Ding, J. , Li, X. , Sykes, B.D. , Rainey, J.K. , and Fliegel, L. 2008. Structural and functional characterization of transmembrane segment IX of the NHE1 isoform of the Na+/H +exchanger. J. Biol. Chem. 283:22018‐22030.
   Roosild, T.P. , Greenwald, J. , Vega, M. , Castronovo, S. , Riek, R. , and Choe, S. 2005. NMR structure of Mistic, a membrane‐integrating protein for membrane protein expression. Science 307:1317‐1321.
   Rost, B. , Sander, C. , Casadio, R. , and Fariselli, P. 1995. Transmembrane helices predicted at 95% accuracy. Protein Sci. 4:521‐533.
   Routzahn, K.M. and Waugh, D.S. 2002. Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins. J. Struct. Funct. Genomics 2:83‐92.
   Schägger, H. 2006. Tricine–SDS‐PAGE. Nat. Protoc. 1:16‐22.
   Shendure, J.A. , Porreca, G.J. , Church, G.M. , Gardner, A.F. , Hendrickson, C.L. , Kieleczawa, J. , and Slatko, B.E. 2011. Overview of DNA sequencing strategies. Curr. Protoc. Mol. Biol. 96:7.1.1–7.1.23.
   Simmerman, H.K. , Collins, J.H. , Theibert, J.L. , Wegener, A.D. , and Jones, L.R. 1986. Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains. J. Biol. Chem. 261:13333‐13341.
   Singh, S.M. and Panda, A.K. 2005. Solubilization and refolding of bacterial inclusion body proteins. J. Biosci. Bioeng. 99:303‐310.
   Slepkov, E. and Fliegel, L. 2002. Structure and function of the NHE1 isoform of the Na+/H+ exchanger. Biochem. Cell Biol. 80:499‐508.
   Slepkov, E.R. , Rainey, J.K. , Li, X. , Liu, Y. , Cheng, F.J. , Lindhout, D.A. , Sykes, B.D. , and Fliegel, L. 2005. Structural and functional characterization of transmembrane segment IV of the NHE1 isoform of the Na+/H+ exchanger. J. Biol. Chem. 280:17863‐17872.
   Tang, X.‐B. , Fujinaga, J. , Kopito, R. , and Casey, J.R. 1998. Topology of the region surrounding Glu681 of human AE1 protein, the erythrocyte anion exchanger. J. Biol. Chem. 273:22545‐22553.
   Trometer, C. and Falson, P. 2010. Mammalian membrane protein expression in baculovirus‐infected insect cells. Methods Mol. Biol. 601:105‐117.
   Tusnády, G.E. and Simon, I. 2001. The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849‐850.
   Tusnády, G.E. , Dosztányi, Z. , and Simon, I. 2004. Transmembrane proteins in the Protein Data Bank: Identification and classification. Bioinformatics 20:2964‐2972.
   Tusnády, G.E. , Dosztányi, Z. , and Simon, I. 2005. PDB:TM: Selection and membrane localization of transmembrane proteins in the protein data bank. Nucl. Acids Res. 33:D275‐278.
   Tzeng, J. , Lee, B.L. , Sykes, B.D. , and Fliegel, L. 2010. Structural and functional analysis of transmembrane segment VI of the NHE1 isoform of the Na+/H+ exchanger. J. Biol. Chem. 285:36656‐36665.
   Wakabayashi, S. , Pang, T. , Su, X. , and Shigekawa, M. 2000. A novel topology model of the human Na(+)/H(+) exchanger isoform 1. J. Biol. Chem. 275:7942‐7949.
   Wallin, E. and Von Heijne, G. 1998. Genome‐wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 7:1029‐1038.
   Wawrzynow, A. , Theibert, J.L. , Murphy, C. , Jona, I. , Martonosi, A. , and Collins, J. H. 1992. Sarcolipin, the “proteolipid” of skeletal muscle sarcoplasmic reticulum, is a unique, amphipathic, 31‐residue peptide. Arch. Biochem. Biophys. 298:620‐623.
   Yeagle, P.L. , Alderfer, J.L. , and Albert, A.D. 1995a. Structure of the carboxy‐terminal domain of bovine rhodopsin. Nat. Struct. Biol. 2:832‐834.
   Yeagle, P.L. , Alderfer, J.L. , and Albert, A.D. 1995b. Structure of the third cytoplasmic loop of bovine rhodopsin. Biochemistry 34:14621‐14625.
   Yeagle, P.L. , Alderfer, J.L. , and Albert, A.D. 1996. Structure determination of the fourth cytoplasmic loop and carboxyl terminal domain of bovine rhodopsin. Mol. Vision 2:12.
   Yeagle, P.L. , Alderfer, J.L. , Salloum, A.C. , Ali, L. , and Albert, A.D. 1997. The first and second cytoplasmic loops of the G‐protein receptor, rhodopsin, independently form beta‐turns. Biochemistry 36:3864‐3869.
   Yeagle, P.L. , Danis, C. , Choi, G. , Alderfer, J.L. , and Albert, A.D. 2000. Three dimensional structure of the seventh transmembrane helical domain of the G‐protein receptor, rhodopsin. Mol. Vision 6:125‐131.
   Zuo, X. , Li, S. , Hall, J. , Mattern, M.R. , Tran, H. , Shoo, J. , Tan, R. , Weiss, S.R. , and Butt, T.R. 2005. Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli . J. Struct. Funct. Genomics 6:103‐111.
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