Dynamics of protein-membrane interactions in drug metabolism and cancer

The cell membrane forms a critical barrier between the environment and the inner biochemical machinery of every life form on earth. Without the membrane, vital metabolic processes almost certainly could not occur. Many of these processes also require proteins present at or even embedded within the membrane. Energy transduction, drug metabolism, and signaling represent some of these dynamic processes which require proteins to interact with cell membranes. One must therefore study protein-membrane interactions deeply to fully understand how organisms function. For example, the orientation of certain protein domains on membranes might impact substrate access, lipid binding, and effector engagement. This highlights the fact that physical parameters such as orientation and binding can influence phenotypes in living systems. These biophysical interactions between proteins and membranes are, regrettably, challenging to study experimentally. Proteins which associate with membranes in cells often irreversibly aggregate upon isolation from the membrane, making them difficult to study with traditional biochemical and biophysical techniques. Detergents are often used to characterize membrane proteins by themselves, but a true membrane is obviously required to study protein-membrane interactions properly. Phospholipid Nanodiscs provide an excellent solution to this problem, and they allow one to control protein and lipid stoichiometry with precision. In this dissertation, I will apply Nanodisc technology to explore physiologically relevant interactions between proteins and membranes. These include the membrane-bound orientation of various cytochromes P450 and the KRas4b oncoprotein. Experimental measurements of the orientation of P450 enzymes provide insight into how transmembrane helix mutations might modulate effector interactions, and a molecular dynamics study of homotropic cooperativity brings atomic-scale mechanistic insight to P450 drug metabolism. Orientation measurements of KRas4b will also be described along with a discussion of future approaches which include more dynamic, time-resolved measurements. Additionally, I will detail new insight into the Nanodisc self-assembly reaction which sets the stage for future work to improve the yield of functional protein in nanodiscs. Overall this work showcases several important advances in our understanding of membrane biology and membrane protein interactions.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Quant Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Camp, Tyler

Contributors dc:contributor

• Sligar, Stephen
• Zhang, Kai
• Selvin, Paul
• Gennis, Robert

• Nanodiscs
• fluorescence
• membranes
• spectroscopy
• orientation

Statement dc:rights

• Copyright 2021 Tyler Camp

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/113035

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/113035