Although the number of membrane protein structures determined to date is relatively low, recent advances in technology such as Synchrotron sources for X-ray crystallography, high field NMR and high resolution electron microscopy have led to increased knowledge in the area of membrane protein biochemistry. Each technique presents its own set of advantages and disadvantages.
1.3.1
X-ray crystallography
X-ray crystallography is currently the most popular method for obtaining the three-
dimensional structure of membrane proteins, accounting for over 80% of all membrane protein structures deposited at the PDB (Berman, Westbrook et al. 2000; Raman, Cherezov et al. 2006). Structures obtained by X-ray crystallography are typically of high resolution, much more so than of those obtained using other structure determination methods, although a prerequisite is often the requirement of diffraction quality crystals that can be hard to grow and often require screening of a large number conditions in order to find those in which crystal growth occurs. Analysis of membrane proteins using X-ray crystallography also presents difficulties as preparation of crystals requires the solubilisation of membrane proteins prior to crystallisation, whilst maintaining the structural integrity of the solubilised protein. Typically membrane proteins are solubilised in detergent, but the
P a g e| 13 presence of detergent when crystallising can prevent the formation of crystal contacts and can also result in distorted structures due to curvature stress induced by the small diameter of detergent micelles. X-ray crystallography is also more suited to larger multi-spanning membrane proteins as larger proteins crystallise more readily than smaller proteins, as the larger the protein the greater the surface area for which crystal contacts can form that are required for crystal growth. For smaller proteins the surface area is greatly reduced thereby reducing the possibility of forming electrostatic contacts between unit cells in a crystal thereby reducing the possibility of forming diffraction quality crystals for analysis, although in recent years the crystallography of membrane proteins in lipid membranes has become viable by the growth of crystals in lipidic mesophases (also referred to as Lipid cubic phase (LCP) crystallisation (Landau and Rosenbusch 1996; Cherezov 2011; Caffrey, Li et al. 2012). Lipids in the LCP form highly curved bilayers that form cubic lattice structures. First used to obtain high resolution structural data for bacteriorhopdsin (Landau and Rosenbusch 1996) this method has now been used to crystallise a variety of bitopic membrane proteins including GPCRs and helical proteins (Cherezov, Rosenbaum et al. 2007; Jaakola, Griffith et al. 2008; Wu, Chien et al. 2010). Therefore whilst X-ray crystallography is positioned to provide high resolution structures at atomic resolution, the strategies involved in producing viable crystallisation conditions can often result in structures that differ vastly from their native form (Cross, Arseniev et al. 1999).
1.3.2
Solution NMR
Next to X-ray crystallography, solution NMR is the second most popular method for the determination of membrane protein three-dimensional structure. Solution NMR techniques allow the possibility of exploring protein-protein and protein-ligand interactions in a dynamic environment without the need for protein crystals. Unlike X-ray crystallography solution NMR can be used in order to study the dynamics of a protein rather than in a fixed state within a crystal. Typically, membrane proteins are studied in detergent micelles, but this is not ideal as detergent micelles have been identified to cause curvature stress to embedded membrane proteins altering the structure of proteins when compared to bilayer
P a g e| 14 bound samples (Chou, Kaufman et al. 2002). In addition the resolution and sensitivity of the spectra obtained by solution NMR is strongly affected by how fast a molecule tumbles in solution. Due to rapid random tumbling rates of small molecules on the NMR timescale (of typically nano/picoseconds), orientation dependant anisotropic interactions are averaged out to zero resulting in sharp resonances. As the size of the molecule in solution increases as does the rate of tumbling, as such larger molecules therefore have much slower tumbling rates and correspondingly shorter spin-spin (transverse) T2 relaxation times due to
enhanced spin-spin interactions. Shorter T2 relaxation times result in line broadening and
intensity loss as a result in of the reduction in the sensitivity of complicated multi-pulse NMR experiments that often use long delays for the necessary coherence transfer steps between nuclei. Therefore whilst the protein of interest to be studied by NMR may be small, once reconstituted into a detergent micelle or lipid embedded environment, the size of the complex typically becomes much larger, thereby tumbling much more slowly (Watts and Spooner 1991; Marcotte and Auger 2005), and in the case of proteins embedded in lipid vesicles can exceed the size limit (100 kDa) for this technique resulting in severe line broadening and signal intensity loss.
1.3.3
Solid state NMR (ssNMR)
In contrast to solution NMR, solid state NMR (ssNMR) is not restricted by an upper molecular weight size limit, thereby making it a powerful technique for the study of higher molecular weight proteins and of those embedded in lipid environments. Therefore, unlike solution NMR, this allows for the study of membrane proteins in hydrated lipid bilayers, thereby representing a more “native-like” environment and therefore resulting in high resolution structures in more biologically relevant confirmations. Typically high resolution ssNMR spectra have been obtained for microcrystalline or amyloid fibril samples with high structural homogeneity (Bockmann and Meier 2010), whereas spectra obtained in hydrated bilayers are often much broader in comparison. As such ssNMR has become an invaluable tool for obtaining structural information of membrane proteins under physiological conditions (Watts, Burnett et al. 1999) such as Gramicidin (Ketchem, Hu et al. 1993), Influenza
P a g e| 15 M2 (Cady, Mishanina et al. 2009; Luo, Cady et al. 2009) and human Phospholamban (Verardi, Shi et al. 2011).
In spite of the numerous advantages of studying membrane protein structure by ssNMR, a number of disadvantages are also associated with the technique, in particular the resolution of ssNMR spectra recorded in comparison to solution NMR spectra is greatly reduced. Inherently, ssNMR spectra are much harder to interpret and assign as they are much more complicated in their nature when compared to solution NMR spectra as the full effect of orientation-dependant (anisotropic) interactions are still present and observed in the spectra obtained. These anisotropic interactions which are normally averaged out in solution for small rapid tumbling molecules are still present in solid samples, as molecular motions are restricted with rotational correlation times much longer than in solution i.e. nanosecond to seconds. In addition, in solid samples, molecules are simultaneously present in a large number of orientations. The presence of anisotropic interactions in solid samples results in considerable broadening of resonances (typically 0.5 – 2 ppm) in comparison to those recorded in solution NMR spectra, often leading to complicated, difficult to resolve. These anisotropic interactions that are still present in ssNMR experiments are listed below.
1.3.4
Chemical shift anisotropy (CSA)
In solid (powder) samples all molecular orientations are present in random orientations, with random distribution, which gives rise to powder patterns in recorded spectra. These powder patterns arise as a result of each different molecular orientation (with respect to the applied magnetic field B0,) having its own chemical shift, with each
orientation giving rise to its own (sharp) resonance. The overlapping of these individual resonances gives rise to the broad axially symmetrical unresolvable powder patterns typically observed in solid samples.
1.3.5
Dipolar coupling
When spins from individual nuclei come into close contact with each other in a sample, the magnetic field generated by each nucleus can act through-space to have an influence on the spin energy of neighbouring nuclei. Dipolar coupling can occur between
P a g e| 16 nuclei of the same type i.e. homonuclear dipolar coupling between 13C and 13C, or between
nuclei of different atoms i.e. heteronuclear dipolar coupling between 13C and 1H. In ssNMR
dipolar coupling can lead to a detrimental effect on the spectra recorded due to signal broadening and decay of magnetisation through effect such as dipolar truncation (Hodgkinson and Emsley 1999). Dipolar interactions can also be useful for probing distance measurements between nuclei due to its r3 dependence, where r is the inter nuclear
distance, therefore making the strength of the dipolar coupling between nuclei a good measure for the distance between them. Dipolar coupling can and also for signal enhancement through transfer of magnetisation such as through cross polarisation, as described further in Section 1.5.2.
1.3.6
Quadrupolar coupling
For nuclei which have spin greater than ½, these nuclei are referred to a quadrupolar, i.e. they possess a nuclear electronic quadrupole moment. The quadrupole moment in the nucleus arises from the non-spherical distribution of charge within. This quadrupole moment is, in addition to the magnetic dipolar moment as possessed by nuclei with spin ½. Electric quadrupoles interact with electric field gradients therefore such nuclei not only interact with the applied and local magnetic fields, but also with any electric field gradients within the nucleus, thereby affecting the nuclear spin energy levels. The strength of the interaction depends upon the magnitude of the quadrupole moment. The effect of the quadrupolar interaction is observed as a substantial broadening of the observed ssNMR spectra.
Therefore, whilst the broad lines in ssNMR spectra contain a wealth of information regarding structure and dynamics of the protein (Warschawski, Traikia et al. 1998), they typically have detrimental effects on spectra recorded, obscuring peaks and leading to the poor resolution of resonances observed, making it difficult to resolve individual resonances due to spectral overcrowding. Therefore in order to avoid spectral crowding, a number of elaborate labelling schemes can employed, such as the use of 1,3-13C labelled glycerol when
expressing membrane proteins, that give rise to specific cross peak patterns that make the assignment process easier.
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