Characterization of Disordered Biomolecular Systems with Scattering Techniques: A Flexible Protein Complex and Solid-Supported Lipid Bilayers

Disorder serves a functional role in cellular membranes and flexible protein domains. An in-plane fluid mixture of phospholipids, carbohydrates, membrane proteins, and

more constitute the plasma membrane. Disordered regions of proteins leverage a wide conformational space to facilitate complexation, signaling, and regulatory interactions.

Experimental approaches which quantify structural information of these systems ideally facilitate biomimetic sample environments. Scattering techniques provide a minimally restrictive platform for characterizing macromolecular systems including lipid bilayers and intrinsically disordered proteins. This thesis contains studies of these systems which utilize X-ray and neutron scattering methods complemented by computational and theoretical structural predictions. The small GTPase KRas acts as a binary switch at the plasma membrane, anchored by a flexible domain, participating in signaling pathways vital for cellular survival and proliferation. Its localization to the plasma membrane is dependent on chaperone proteins, such as SmgGDS, which bind and traffic it through the cytosol. Two isoforms of SmgGDS regulate the prenylation and localization of small GTPases: SmgGDS-607

binds unprenylated small GTPases while SmgGDS-558 associates and houses the lipid anchor of prenylated small GTPases in a hydrophobic pocket. Structural and thermodynamic details of KRas complexation with SmgGDS are critical for understanding Ras biology and for identifying potential targets for chemical inhibition of oncogenic KRas signaling. We show that SmgGDS-558 readily solubilizes KRas bound to anionic membranes with a binding affinity larger than that of KRas associating with the membrane.

Using a combination of solution scattering and molecular dynamics simulations, a configurational ensemble of a flexible SmgGDS-558/KRas complex was determined

in which specific interactions were found to be limited to the C-terminal end of the hypervariable region. Informed by mutational studies which identify a collection of

SmgGDS residues as important for binding KRas, we observe a set of anionic residues near the hydrophobic pocket of SmgGDS-558 directly associating KRas within the

determined flexible ensemble. Structural studies of membrane proteins frequently utilize model membranes supported by solid-state surfaces. Understanding fundamental interactions between lipid membranes and such surfaces is crucial, not only for the development of novel architectures for characterizing membrane proteins, but also for conceptualization of biosensors which detect and transduce stimuli at the bilayer-substrate interface. We have developed an experimental and theoretical framework for tuning the association of zwitterionic lipid bilayers with solid substrates. In this effort, we performed systematic neutron reflectometry studies which identify the formation mechanism of a novel, facile technique for synthesizing solid-supported membranes. We observed headgroups of a lipid monolayer associating with the surface in hydrophobic solvent, which form a template for subsequent self-assembly of the lipid bilayer on exchange with aqueous solvent.

We fabricated complete membranes at surface chemistries unamenable to conventional bilayer formation methods such as vesicle fusion. A mean-field approach to modeling

the free energy of bilayer-substrate interactions as a superposition of electrostatic, van der Waals, steric, and short-range hydration components was assessed with the modeled interfacial structure derived from neutron reflectometry data. The resulting free energy model of this experimental system quantitatively predicts the dependence of the lipid membrane separation distance on the substrate surface charge. By altering substrate surface chemistry and aqueous buffer composition, the range and magnitude

of electrostatic forces can be finely tuned relative to the van der Waals, confinement, and hydration forces which are invariant to these electrochemical properties. In this

way, the morphology of a “floating” bilayer was finely controlled at the nanoscale-level out to a separation distance of 40 °A where out-of-plane bilayer undulations are weakly suppressed.