Conformational changes induced by fatty acids

Previous studies indicate that a conformational change occurs when fatty

acids bind to HSA (Curry et al., 1998; Bhattacharya et al., 2000). This causes the relative movement between domains I and II and results in

disruption of the Zn2+ binding site as His67/Asn99 and His247/Asp249 are

no longer in close proximity to each other (see Figure 1.5). Previously,

conformational changes induced by fatty acid binding have been studied

by other approaches including cross linking of Lys residues followed by

tryptic digestion and analysis of the peptides produced (Huang et al.,

2005). TWIM-MS was used to compare the mobility of HSA samples that

102 myristate present. The HSA was studied in the presence of myristate and

not myristate/Zn2+ so the experimental cross section could be directly

compared to the theoretical value calculated from the structure deposited

in the PDB. Although the Synapt used a much gentler nanospray source,

a HSA-myristate complex could not be preserved as no significant

increase in mass was observed (cf. Chapter 3). However, HSA in the

presence myristate showed an increased collisional cross section

compared to apo-HSA under the same conditions. The sample that had

been incubated with myristate had a large increase in cross section which

was estimated to be 87 Å2 for the 15+ charge state (Figure 4.7).

Figure 4.7 Dependence of charge state for the estimated collisional cross sections of apo-HSA and HSA in the presence of 5 mol. equiv. myristate. There are large differences in cross sections observed for each protein sample.

In order to validate these experimental data, theoretical cross sections

were calculated using MOBCAL, an open source program (Mesleh et al.,

1996; Shvartsburg and Jarrold, 1996). X-ray crystal structures are available for apo-HSA and myristate bound HSA, files 1AO6 and 1BJ5 in

103 the Protein Data Bank, respectively. Theoretical cross sections are

calculated based on projection approximation (PA) and exact hard sphere

scattering (EHSS) approximation. The PA method can underestimate the cross sections of more complex molecules such as proteins as they undergo more interactions with the buffer gas. The EHSS method takes into account the scattering between the ion and the buffer gas correctly but can overestimate cross sections (Jarrold, 1999). However, these models are used because they take substantially less computational time than others such as the more accurate trajectory method (TJ) (Jarrold, 1999).

The PA and EHSS cross sections for 1AO6 and 1BJ5 are outlined in Figure 4.8 B. The experimentally-determined cross sections fall between the theoretical values for the PA and EHSS. There is good agreement between the experimental values and those calculated from the PA method but the EHSS method overestimates the cross section of HSA significantly. Some examples of native gas phase conformations have smaller estimated cross sections than those obtained from the EHSS method, for example in the case of bovine pancreatic trypsin inhibitor (BPTI) (Shelimov et al., 1997). This could be due to a lack of water molecules which would otherwise occupy cavities in the protein structure and increase its size. Furthermore, polar side chains, which would usually extend out into the solvent, collapse onto the protein surface in the gas phase (Shelimov et al., 1997).

104

Complex HSA (1AO6) HSA-5Myr (1BJ5) Experimental 3920 Ų 4007 Ų

PA 3847 Ų 3923 Ų

EHSS 5054 Ų 5127 Ų

Figure 4.8 Comparison of experimental and theoretical collisional cross sections for HSA complexes. A) Overlay of the protein structures from 1AO6 (red; FA free) and 1BJ5 (green; 5 myristates) B) Table summarising the experimental cross sections and those obtained from the PA and EHSS methods.

The theoretical difference between the cross sections calculated from the PA method is 76 Å2 which compares well with the experimental difference of 87 Å2. An overlay of 1AO6 and 1BJ5 is also shown in Figure 4.8 A which indicates the structural changes induced by fatty acid binding. The crevice in the centre of the protein is considerably expanded and domains I and III are moved away from each other.

Taking into account the experimental and theoretical results, it can be

suggested that even though the interactions with fatty acids were broken

on entering the gas phase, the expanded conformation induced by fatty-

acid binding in solution was apparently retained in the gas phase during

the time in which the experiment was performed. This can be rationalised

B

A

105 as it has been shown that gas phase structures reflect the native

structure of proteins in solution over short time scales (Hoaglund-Hyzer,

1991; Breuker and McLafferty, 2008). A study of cytochrome c that

involved trapping the ions for various periods of time prior to IM

experiments showed that protein native structure was retained for 30-60

ms. Trapping the ions for longer than this resulted in unfolded

conformations being observed (Badman et al., 2005). Other reports have

indicated that structural features can be retained for longer than 100 ms

(Wyttenbach et al., 2009). The TWIM-MS experiments in this work were

carried out on a shorter timescale of 10-20 ms therefore there is less

chance for the solution structure to rearrange into the most stable FA-

free conformation in the gas phase.