Probing environmental effects on gas-phase protein structure

Thesis

Growth in the use of mass spectrometry as an analytical tool for studying protein structure and functionality has posed questions regarding correlation of native structures and observed gas-phase conformations for proteins. This thesis explores the relationship between the solution-phase and gas-phase structures of soluble proteins, and investigates the effect of the lipid environment on membrane proteins.

To assess gas-phase structure, ion-mobility mass spectrometry was used to measure directly the collision cross-sections of soluble proteins across a broad mass range. These collision cross-sections values were then compared with those derived from solution-phase data, calculated from dynamic light scattering experiments. Using computational methods collision cross-sections were calculated from structures reported in the protein data bank using a variety of techniques. Differing physical environments in solution and gas phases are not shown to affect protein structure by the dynamic light scattering and ion-mobility measurements. Contrastingly the computational calculations demonstrate that there is less correspondence between x-ray structures and their gas-phase counterparts.

Despite the effect that lipid environment has on membrane protein structure, capturing this native environment has been challenging. Lipodisqs look to overcome this problem by removing the portion of the membrane surrounding the embedded protein. Lipodisqs were investigated as potential vehicles for membrane protein mass spectrometry and compared with conventional detergent based approaches. Lower charge states are observed for proteins released from lipodisqs, compared to those released from detergent micelles, increasing the probability of native features being retained including native-like protein-lipid interactions. Lipodisqs therefore demonstrate significant potential as a tool for membrane protein mass spectrometry.

In summary, through this body of experimental work relationships have been established between gas-phase and solution-phase structures as well as computational methods. The use of Lipodisqs to eject membrane proteins from regions of the native membrane has been investigated.

Authors: Hewitt, D

• DOI: 10.5287/ora-m82xzgbnj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English