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Approaches for Preparation and Biophysical Characterization of Transmembrane β-Barrels

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Chemical and Synthetic Approaches in Membrane Biology

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

Membrane proteins are influential members of the cellular machinery, as they are involved in a range of critical functions from immune receptors and ion transporters to enzymes and signaling molecules. These facets render membrane proteins as ideal drug targets. Despite their significance in biological systems, our knowledge of the structural and functional behavior of membrane proteins remains considerably limited, when compared with their soluble counterparts. This limitation continues to persist, owing largely to the difficulties in membrane protein purification and amenable methods for their characterization. In this review, we outline the methods for the extraction and purification of transmembrane β-barrel proteins, which will serve as a guide for generating membrane protein preparations in high amounts and of sufficient purity. Next, we provide a detailed description of the currently available protocols for efficient refolding of β-barrel membrane proteins in both lipidic and detergent systems. These protocols are designed to enable the protein to attain its native structural and functional characteristics in any chosen membrane mimetic environment. Further, we detail the current methodologies being employed for the biophysical and structural characterization of transmembrane β-barrels, using illustrative examples drawn from recent studies using various β-barrel membrane proteins. This consolidated summary of protocols and strategies will prove highly useful to membrane biologists and can be applied to the study of any uncharacterized β-barrel membrane protein.

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Abbreviations

β-OG:

n-Octyl-β-d-glucoside

τ c :

Rotational correlation time

BLM:

Bilayer lipid membrane

CD:

Circular dichroism

D7PC:

1,2-Diheptanoyl-sn-glycero-3-phosphocholine

DDM:

n-Dodecyl β-d-maltoside

DHPC:

1,2-Dihexanoyl-sn-glycero-3-phosphocholine

DLPC:

1,2-Dilauroyl-sn-glycero-3-phosphocholine

DMPC:

1,2-Dimyristoyl-sn-glycero-3-phosphocholine

DPC:

n-Dodecylphosphocholine

DPR:

Detergent-to-protein ratio

FRET:

Förster resonance energy transfer

HSQC:

Heteronuclear single quantum coherence

IB:

Inclusion bodies

IEX:

Ion-exchange chromatography

k q :

Bimolecular quenching constant

K SV :

Stern-Volmer constant

LDAO:

Lauryldimethylamine oxide

LPR:

Lipid-protein ratio

MP:

Membrane protein

MS:

Mass spectrometry

NMR:

Nuclear magnetic resonance

<t>:

Average fluorescence lifetime

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Acknowledgments

B.R.I. thanks the University Grants Commission, Govt. of India, for Senior Research Fellowship. A.G. thanks IISER Bhopal for Senior Research Fellowship. R.M. is a Wellcome Trust/DBT India Alliance Intermediate Fellow. This work is supported by the Wellcome Trust/DBT India Alliance award number IA/I/14/1/501305, Department of Biotechnology, Govt. of India award numbers BT/01/IYBA/2009 and BT/HRD/35/02/2006, and the Science and Engineering Research Board of the Department of Science and Technology, Govt. of India award number SR/FT/LS-47/2010 to R.M.

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    1. Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Academic Building III, Bhopal Bypass Road, Bhauri, Bhopal, 462066, India

      Bharat Ramasubramanian Iyer & Ankit Gupta

    2. Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Academic Building III, Room 324, Bhopal Bypass Road, Bhauri, Bhopal, 462066, India

      Radhakrishnan Mahalakshmi

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    1. Bharat Ramasubramanian Iyer
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    2. Ankit Gupta
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    Correspondence to Radhakrishnan Mahalakshmi .

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    1. Indian Institute of Technology, Kanpur, India

      Arun K. Shukla

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    Iyer, B.R., Gupta, A., Mahalakshmi, R. (2016). Approaches for Preparation and Biophysical Characterization of Transmembrane β-Barrels. In: Shukla, A. (eds) Chemical and Synthetic Approaches in Membrane Biology. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/8623_2016_4

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    • DOI: https://doi.org/10.1007/8623_2016_4

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