Membrane proteins are coded by up to 30% of the open reading frames in know genomes. They have pivotal roles in many biological processes including: transport of ions and molecules, control of transmembrane potential, generation and transduction of energy, signal recognition and transduction, cell-cell communication, enzymatic activity, structural roles. Mutations in membrane proteins are linked with various human diseases including: Alzheimer’s disease, Brugada syndrome, cancer, cystic fibrosis, heart disease, hypothyroidism, lysosomal storage disease, nephrogenic diabetes insipidus, retinitis pigmentosa. Membrane proteins are the molecular targets for around 50-60% of validated drugs and they remain a principal target for new drug discovery. Despite all this, the number of structures of membrane proteins is less than 1% of total protein structures in the Protein Data Bank due to various challenges associated with applying the main biophysical techniques for high-resolution protein structure determination: X-ray crystallography, electron microscopy, NMR spectroscopy. There is an infinite amount of information and understanding yet to be obtained about the structure, function and molecular mechanism of membrane proteins and their ligands.
This book “A Closer Look at Membrane Proteins” brings together recent developments in the structures, molecular mechanisms and roles of some different types of membrane proteins using various computational and experimental methods, and also views on the challenges around expression and purification of membrane proteins and a successful demonstration of how these challenges can be overcome.
Chapter One considers insulin-like growth factor receptors and their roles in initiating mitogenic and metabolic pathways involved in cell growth and proliferation and energy metabolism, and also their roles in cell apoptosis. Information on the receptors is related to normal and abnormal tissue growth and development, using placental and colorectal tissues as examples. Chapter Two demonstrates how transmembrane protein transport across the nuclear envelope can be imaged at high-resolution using dynamic single-molecule microscopy; especially how the technique can be used to interrogate different proposed models for the mechanism of membrane protein transport: diffusion-retention, ATPdependent, nuclear localization signal–mediated, sorting motif–mediated.
Computer simulation provides a way to study the structure and function of membrane proteins, alternative to using laboratory techniques, and this is the subject of Chapter Three. The focus is on large scale molecular dynamics (MD) simulations with special emphasis on scalable parallel methods, and how correctly relating molecular structures to the physiological properties of proteins is a major challenge in the field. Chapter Four consolidates general principles of secondary active transporter function, which catalyse transport of ions and small molecules across cell membranes against electrochemical gradients. It considers thermodynamics and molecular mechanism and how these transporters cycle between inward- and outward-facing conformations. Also how experimental structural data and MD simulations indicate that transporters can be understood as gated pores. A unified picture emerges in which symporter, antiporter and uniporter function are extremes in a continuum of functionality.
Following recent high-resolution X-ray crystal structures of substrate-bound proteins, Chapter Five reviews emerging structural insights about multidrug recognition and extrusion by MATE (Multidrug and Toxic Compound Extrusion) and MFS (Major Facilitator Superfamily) secondary active transporters, which provide a mechanism of resistance to therapeutic drugs. In addition to providing a better understanding about the underlying mechanism of multidrug extrusion, this chapter engenders new ideas about how to curtail efflux-mediated multidrug resistance. A myriad of membrane proteins in the pathogenic bacterium Vibrio cholerae are described in Chapter Six that contribute to its physiology, virulence and antimicrobial resistance. These include outer membrane proteins and efflux pumps of the RND (Resistance-Nodulation-Division) family and MFS. The chapter emphasises how inhibition of efflux pumps can reduce virulence of V. cholerae and restore susceptibility to conventional antibiotics, and demonstrates how a complex network involving quorum sensing, efflux pumps and virulence gene expression regulates physiology and virulence.
The challenges around expression and purification of integral membrane proteins and performing laboratory experiments to study their structure and function are well recognised. In this respect, Chapter Seven gives a personal view on “The commandments of studying integral membrane proteins”. These commandments consider integral membrane protein expression and purification, biochemistry, functionality studies and high-resolution structures. It is possible to overcome the challenges for expression and purification of integral membrane proteins, especially by those who are suitably experienced and have longevity of success. This is demonstrated in Chapter Eight by the amplified expression, functional characterisation and purification of a cytosine transporter of the NCS1 (Nucleobase Cation Symporter-1) family from the bacterium Vibrio parahaemolyticus. The gene was cloned into plasmid pTTQ18 along with a sequence for introducing a C-terminal hexahistidine-tag to aid purification and amplified expression achieved in Escherichia coli BL21(DE3). The secondary structure and stability of the purified protein was analysed by circular dichroism spectroscopy and the protein was confirmed as a cytosine transporter by radiolabelled transport measurements in whole cells.
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