Measuring CPT1B TM1 – TM2 Interactions Using CD Spectroscopy

Once the alpha helicity of both CPT1B TM1 and TM2 had been confirmed in DPC micelles, CD spectroscopy was used as a method for detecting TM1 – TM2 interactions. This could be achieved, either through an enhancement in their helicity on interaction, or if a coiled coil structure is formed this can be observed. This approach has been used in the past to study synthetic peptides of the kinesin neck region (Tripet, et al., 1997). Sections of the protein were analysed for secondary structure and if the two helical peptides interact through a coiled coil interaction, the ratio of the two negative alpha helical signals is altered. A 222:208 nm ratio >1 indicates coiled coil formation, and <1 indicates a non-coiled coil alpha helical structure or a monomer (Tripet, et al., 1997).

A sample was prepared containing 25 µM each of CPT1B TM1 and TM2 (to maintain a total peptide concentration of 50 µM) in 100 mM DPC. A CD spectrum was measured and then compared against one arithmetically produced by the addition of the individual CPT1B TM1 and TM2 spectra (results shown in Figure 6.5). If no interactions between the two peptides occurred then the experimental spectrum should match closely with the arithmetically produced spectrum. If interactions between the two peptides were occurring then a significant difference between the two spectra was expected.

Figure 6.5 – Measuring CPT1B TM interactions using CD.Calculated arithmetic addition of the CPT1B TM1 and TM2 spectra recorded before (black) and the measured experimental result on mixing the two peptides (red).

A difference was observed between the arithmetically added CPT1B peptide spectra, and that resulting from the mixture of the two. The change in the signal however was unexpected. There was a loss in the characteristic alpha helical minimum at 208 nm entirely and a shift of the minimum at 222 nm. Despite this surprising result, it does show that there is a change upon addition of CPT1B TM2 to

TM1 indicating that the two peptides are interacting, and so this suggests that CD spectroscopy can be used to detect CPT1B TM domain interactions in DPC micelles. More experiments, including titration experiments in which one of the TM domain peptides would be slowly added to the other, were planned in order to measure this effect more closely, however a shortage of materials prevented these experiments.

6.3 NMR

Due to the difficulties in crystallising membrane proteins, NMR spectroscopy is a valuable tool in the study of their structure. Many soluble protein structures have been solved using NMR spectroscopy, however due to the hydrophobicity of membrane proteins a membrane mimetic is required to stabilise the native fold of these proteins. This membrane mimetic is often a detergent micelle used to solubilise the hydrophobic parts of the protein. This is advantageous in presenting a native membrane-like environment to the membrane protein but also significantly increases the size of the protein-micelle complex to be studied. This increase in size makes the study of even relatively small membrane proteins more difficult by NMR.

NMR spectroscopy has been used to calculate structures and measure oligomerisation in isolated TM domain sections in proteins previously (Bocharov, et al., 2012). These studies have relied on being able to assign NMR signals to specific amino acids in the protein sequence. Due to a reduction in expression yield when including both isotopic labels, the peptides expressed and purified inChapter 5were only 15N labelled so a three dimensional sequential assignment using HNCA and HN(CO)CA type experiments was not possible as these rely on 13_{C labelling as well}

spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) can however be used to assign small proteins and peptides by assigning the amino acid side chains, and this was the strategy to be employed here.

In addition to 1H homonuclear experiments, 15N labelling allows 1H-15N heteronuclear experiments such as HSQC to be performed. HSQC experiments are useful in providing a fingerprint of a protein. An HSQC spectrum records all NH correlations in a protein which means a peak is observed for each amide group in the protein backbone as well as any NH groups in the amino acid side chains. As a signal is observed for each non-proline amino acid in an HSQC spectrum, this makes HSQC experiments an excellent method for detecting changes in the environment of a protein. If the HSQC can be assigned, then any perturbations in the chemical shift of these peaks can be directly mapped back to a specific amino acid. This method was used to investigate the interactions between the CPT1B TM1 and TM2 peptides; increasing amounts of CPT1B TM2 was added to a sample of CPT1B TM1 and chemical shift perturbations in the HSQC spectrum were recorded. Here experiments are presented to assess purity of the expressed peptides (Chapter 5) as well as to assign individual residues and study TM domain interactions in membrane mimicking detergent micelles.