Zachary A. Levine, PhD
Postdoctoral Researcher | Shea Lab
Departments of Physics, Chemistry and Biochemistry
Materials Research Laboratory
University of California, Santa Barbara
Research Experience:

Postdoctoral research:
Departments of Physics, Chemistry & Biochemistry, and the Materials Research Laboratory – UC Santa Barbara (2013-Present)
My current research involves studying the folding and aggregation of intrinsically-disordered proteins (IDPs) implicated in human degenerative diseases using enhanced-sampling molecular dynamics simulations. Protein function is often deduced from its structure, however IDPs exhibit a wide variety of transient structures that are difficult to characterize experimentally. Atomistic simulations, combined with parallel-tempering samping, allows for the analysis of IDP ensembles, where free energy landscapes can be extracted that describe populations of proteins rather than single molecules. This data allows for multiple protein folding pathways to be identified in amyloids and other aggregating proteins implicated in human diseases such as Alzheimer’s Disease, Parkinson’s Disease, Type II Diabetes, and other amyloid diseases. By better understanding precisely what physical and chemical factors affect IDP folding and aggregation, especially in the context of complex physiological environments such as biological surfaces, we can predict how proteins implicated in disease become pathologically dysregulated, and perhaps design interventions to counteract such effects.

Selected Topics:

Regulation and Aggregation of Intrinsically Disordered Peptides

Intrinsically disordered proteins (IDPs) are a unique class of proteins which have no stable native structures, a feature which allows them to adopt a wide variety of extended and compact conformations that facilitate a large number of vital physiological functions. One of the most well-known IDPs is the microtubule associate tau protein which regulates microtubule growth and shortening in the human nervous system. However, dysfunctions in tau can lead to tau oligomerization, fibril formation, and neurodegenerative disease, including Alzheimer’s disease. Using a combination of experiments and simulations, we explored the role of osmolytes in regulating the conformation and aggregation propensities of the R2/wt peptide, a fragment of tau containing the aggregating PHF6* sequence VQIINK. We showed that the osmolytes urea and TMAO shift the population of IDP monomer structures, but that no new conformational ensembles emerge. While urea halts aggregation, TMAO promotes the formation of compact oligomers (including helical oligomers) through a newly proposed mechanism of redistribution of water around the perimeter of the peptide. We put forth a superposition of ensembles hypothesis to rationalize the mechanism by which IDP structure and aggregation is regulated in the cell.

Surface Force Measurements and Simulations of Mussel-Derived Peptide Adhesives on Wet Organic Surfaces

The need for bio-inspired wet adhesives has significantly increased in the past few decades (e.g., for dental and medical transplants, coronary artery coatings, cell encapsulants, etc.). However, the molecular basis behind catechol-facilitated adhesion to organic surfaces remains unclear, thus hindering synthesis and optimization of novel underwater adhesives. The present combined experimental and theoretical study reconciles bioadhesion measurements of novel catechol-containing peptides to self-assembled monolayers (SAMs) with all-atom molecular dynamics simulations, yielding a comprehensive framework that explicitly identifies the basis for underwater adhesion. Simulations and surface forces apparatus measurements agree with one another, and both approaches show strong peptide adhesion to hydrophobic SAMs, and weak peptide adhesion to hydrophilic SAMs, providing a starting point for the development of next-generation underwater glues.

To What Extent Does Surface Hydrophobicity Dictate Peptide Folding and Stability near Surfaces?

Protein-surface interactions are ubiquitous in both the cellular setting and in modern bioengineering devices, but how such interactions impact protein stability is not well understood. We investigate the folding of the GB1 hairpin peptide in the presence of self-assembled monolayers and graphite like surfaces using replica exchange molecular dynamics simulations. By varying surface hydrophobicity, and decoupling direct protein–surface interactions from water-mediated interactions, we show that surface wettability plays a surprisingly minor role in dictating protein stability. For both the beta-hairpin GB1 and the helical miniprotein TrpCage, adsorption and stability is largely dictated by the nature of the direct chemical interactions between the protein and the surface. Independent of the surface hydrophobicity profile, strong protein–surface interactions destabilize the folded structure while weak interactions stabilize it.

Trp-Cage Folding on Organic Surfaces: Theoretical Predictions and Comparisons

Trp-cage is an artificial miniprotein that is one of the smallest and most stable self-folding proteins in aqueous environments, due to concerted hydrophobic shielding of a Trp residue by nearby polyproline helices. Simulations have extensively characterized Trp-cage denaturation and found that folding takes place on the order of microseconds, however the interactions of Trp-cage with organic surfaces (e.g. membranes) and their effect on protein conformation is largely unknown. To better understand these interactions we utilized a combination of replica-exchange molecular dynamics (REMD) and metadynamics (MetaD) simulations for 200 ns/replica, to investigate Trp-cage folding on self-assembled monolayers (SAMS) under the AMBER99SB-ILDN and AMBER03 force fields. We found that in both REMD and MetaD systems, Trp-cage strongly binds to zwitterionic CH3 surfaces (-25 kT) and moderately adsorbs to anionic COOH interfaces (-7.6 kT), with Trp hydrophobicity driving CH3 adhesion and electrostatic attractions from Lys and Asn driving COOH adhesion. Similar to solid-state surfaces, SAMs facilitate a number of intermediate Trp-cage conformations between the folded and unfolded state. Furthermore, Trp-cage is also more resistant to temperature denaturation when attached to organic surfaces, despite the fact that CH3 interfaces decrease Trp-cage’s native secondary structure. Regarding Trp-cage’s aromatic groups in zwitterionic CH3 systems, Tyr becomes oriented parallel to the surface in order to maximize hydrophobicity while Trp remains caged perpendicular to the surface, however Trp can reorient itself parallel to the interface as the miniprotein more-closely binds to the surface. In contrast, Tyr and Trp are both repelled from COOH surfaces, though Trp-cage still adheres to the anionic interface electrostatically via Lys and its N-terminated Asn residue. We also observe that the AMBER03/SPCE force field results in more stable Trp-cage structures on the surface of SAMs compared to AMBER99SB-ILDN/TIP3P, with both enhanced sampling methods displaying quantitative similarity across the two force fields.


Determination of Biomembrane Bending Moduli in Fully Atomistic Simulations

The bilayer bending modulus (Kc) is one of the most important physical constants characterizing lipid membranes, but precisely measuring it is a challenge, both experimentally and computationally. Experimental measurements on chemically identical bilayers often differ depending upon the techniques employed, and robust simulation results have previously been limited to coarse-grained models (at varying levels of resolution). This work demonstrated the extraction of Kc from fully atomistic molecular dynamics simulations for three different single-component lipid bilayers (DPPC, DOPC and DOPE). The results agreed quantitatively with experiments that measure thermal shape fluctuations in giant unilamellar vesicles. Lipid tilt, twist, and compression moduli were also reported.


Graduate Research:
Information Sciences Institute/Department of Physics – University of Southern California (2008 - 2013).
My interdisciplinary research at the USC Information Sciences Institute focused on modeling the electropermeabilization of cellular membranes with external electric fields, in order to enhance the uptake of therapeutic molecules (e.g. chemotherapy drugs) or nucleic acids into cells. This work spanned across the departments of physics, computer science, chemistry, and electrical engineering.

Selected Topics:

Electroporating Fields Target Oxidatively Damaged Areas in the Cell Membrane
Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy.

Life Cycle of an Electropore: Field-Dependent and Field- Independent Steps in Pore Creation and Annihilation
Electropermeabilization, an electric field-induced modification of the barrier functions of the cell membrane, is widely used in laboratories and increasingly in the clinic; but the mechanisms and physical structures associated with the electromanipulation of membrane permeability have not been definitively characterized. Indirect experimental observations of electrical conductance and small molecule transport as well as molecular dynamics simulations have led to models in which hydrophilic pores form in phospholipid bilayers with increased probability in the presence of an electric field. Presently available methods do not permit the direct, nanoscale examination of electroporated membranes that would confirm the existence of these structures. To facilitate the reconciliation of poration models with the observed properties of electropermeabilized lipid bilayers and cell membranes, we propose a scheme for characterizing the stages of electropore formation and resealing. This electropore life cycle, based on molecular dynamics simulations of phospholipid bilayers, defines a sequence of discrete steps in the electric field-driven restructuring of the membrane that leads to the formation of a head group-lined, aqueous pore and then, after the field is removed, to the dismantling of the pore and reassembly of the intact bilayer. Utilizing this scheme we can systematically analyze the interactions between the electric field and the bilayer components involved in pore initiation, construction and resealing. We find that the pore creation time depends strongly on the electric field gradient across the membrane interface and that the pore annihilation time is at least weakly dependent on the magnitude of the pore-initiating electric field and, in general, much longer than the pore creation time.

Calcium and Phosphatidylserine Inhibit Lipid Electropore Formation and Reduce Pore Lifetime

Molecular dynamics simulations of electroporation of homogeneous phospholipid bilayers show that the pore creation time is strongly dependent on the magnitude of the applied electric field. Here, we investigated whether heterogeneous bilayers containing phospholipids with zwitterionic and anionic headgroups exhibit a similar dependence. To facilitate this analysis we divide the life cycle of an electropore into several stages, marking the sequence of steps for pore creation and pore annihilation (restoration of the bilayer after removal of the electric field). We also report simulations of calcium binding isotherms and the effects of calcium ions on the electroporation of heterogeneous lipid bilayers. Calcium binding simulations are consistent with experimental data using a 1:2 Langmuir binding isotherm. We find that calcium ions and phosphatidylserine increase pore creation time and decrease pore annihilation time. For all systems tested, pore creation time was inversely proportional to the bilayer internal electric field.





© Zachary A. Levine |