Introduction

3.1.1 Crystallography

A protein’s function in vivo is dependent on the formation of its correct three- dimensional (3D) structure. Often proteins that share similar folds or structural motifs perform similar functions. The elucidation of the structure of a protein, and its comparison to similar structures can provide clues about its function for further biochemical investigations.

There are two main methods used to solve the 3D structure of protein’s; solution NMR and X-ray crystallography. Solution NMR provides not only structural data, but also protein dynamic data that gives information on the proteins movements allowing monitoring of protein ‘breathing’. NMR requires large amounts of isotopically labelled protein, the main disadvantages of NMR are the size constraints that current technology imposes and the large amounts of time required to assign peaks. At 29 kDa, Yih1 is on the upper limit of protein size to be analysed by NMR.

X-ray crystallography captures the protein in a static state and although no direct protein dynamic data is collected, some inferences may be drawn about flexible regions as they often give poor diffraction data. Protein crystallisation requires a highly pure (preferably >99%), homogeneous protein solution and the correct conditions to drive the organisation of those protein molecules into an ordered crystalline lattice. The protein crystal is analysed by X-ray diffraction and the resulting data is processed by various methods to produce an electron density map into which each amino acid in the primary sequence can be modelled.

The main bottleneck in X-ray crystallography is the formation of diffraction quality crystals. The conditions required to form crystals varies from protein to protein and there is no way to predict what conditions are likely to work for any given protein. Proteins which are inherently flexible will generally not form crystals due to conformational heterogeneity.

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3.1.2 Expression and Purification of Yih1

In the study of proteins by crystallography, pure protein is required. Pure Yih1 was obtained by expression in the heterologous host Escherichia coli and purification from the resulting cellular extract by standard biochemical chromatographic techniques.

Various Yih1 constructs were expressed and purified in the attempt to crystallise Yih1. The rationale behind this is that the addition of tags at either the N-terminus or C-terminus of a protein can affect the formation of crystal contacts. Yih1 was expressed without a tag (Yih1), with an N-terminal His6-tag (His6-Yih1), a C-terminal His6-tag (Yih1-His6), or as an N-Terminal His6 tagged mutant where the cysteines of Yih1 had been mutated to alanines (His6-Yih1cys-). His6-Yih1cys- was used to prevent the possible formation of inter- or intra-molecular disulfide bonds involving the two cysteines present in the RWD of Yih1. As Yih1 is a cytosolic protein, it would not be expected to form disulphide bonds. The formation of both intra- and inter-molecular disulphide bonds during extraction and purification therefore has the potential to add to the heterogeneity of the sample, reducing the likelihood of forming an ordered crystalline lattice.

Two purification steps were carried out to separate the tagged and untagged Yih1 from endogenous E. coli proteins. Untagged Yih1 was purified by anion exchange chromatography followed by size exclusion chromatography whereas His6-tagged variants were purified by immobilised metal affinity chromatography again followed by size exclusion chromatography. The rationale behind the different purification methods are outlined below.

Anion exchange chromatography (AEX) utilises the charge differences between various proteins to separate them. The more negatively charged a protein is at the experimental pH, the higher the concentration of Cl- ions are required to displace it from the more positively charged matrix.

Immobilised Metal Affinity Chromatography (IMAC) exploits the properties of divalent metal ions (most commonly Ni2+) that interact with indole-containing compounds. Proteins containing a His-tag (six or more Histidine residues in a row at the N- or C-

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terminus of the protein) bind to the nickel-charged matrix and can be eluted with increasing concentrations of imidazole which competes with the protein for interaction with the matrix.

Size Exclusion chromatography (SEC) separates proteins based on their hydrodynamic/Stokes radius with larger proteins eluting before smaller proteins due to the smaller proteins being able to enter the pores in the resin, more fully retarding their progression through the column.

3.1.3 Biochemical Analysis of Yih1 and Variants

A protein’s expression and solubility are two important factors on the path to forming diffraction quality crystals, but neither guarantees that the protein of interest is folded correctly or reveals if it has any modifications made to it. To tackle these questions, Yih1 and some variants of Yih1 were subjected to analysis by circular dichroism and mass spectrometry.

Far UV Circular Dichroism (CD) is a method that utilises the ability of protein secondary structure to differentially absorb left and right circularly polarised light to

Figure 3.1 Circular Dichroism Standards

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determine structural elements present within a protein. The three main forms of secondary structure, α-helix, β-sheet and random coil, all have CD spectra characterised by specific maxima and minima, and hence specific shapes (see Figure 3.1). Analysis of CD spectra and comparison with a database of measurements from a number of proteins whose structure have been solved by X- ray diffraction allow predictions to be made on the ratios of the different secondary structural elements that are likely to be present. There are various algorithms used to deduce the proportions of secondary structural elements from far UV CD spectra, none of which are 100% accurate. Although Yih1 has been previously analysed by far UV CD and predicted to be composed of 29% helices, 24% β-sheets, 20% turns, and 28% random structure (Sattlegger et al., 2011), the analysis of purified Yih1 by CD is one way to check that the protein being used in crystallisation experiments is folded.

Mass Spectrometry accurately measures the mass-to-charge ratio of components within a sample. It allows the identification of post-translational modifications such as phosphorylation and oxidation that can affect the propensity of a protein sample to crystallise.

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