Professor Xiao Hu, Department of Physics and Astronomy, Rowan University

 RUI: Unraveling the Structure-Activity Relationships of Resilin Biomaterials by Joint Experimental-Computational Approaches

Resilin, known as a “super elastic rubber”, has drawn intensive research interests for its striking energy transduction and storage properties.  However, due to lack of atomic structures of this protein and its constituent domains, no models with atomic details are available up to date to enable an in-depth understanding of the energy transductions and storage mechanism in resilin.  In our current study, with model peptides, we explore the structures of the high-hydrophobicity sequence and the repeats of the Exon III domain of Drosophila melanogaster resilin at the atomic level with combinations of computational and experimental techniques.  Bioinformatics method predicts dominant β-sheet and β-turn structures for the above respective sequences, which is consistent with earlier and present experimental acquisitions.  Molecular dynamics (MD) simulations with the explicit solvent model further confirm these structural features.  The peptide secondary and tertiary structures, and peptide-water interactions are investigated in details with simulations, and the results are compared to experimental measurements from Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichroism (CD).  The present study offers an atomistic view of resilin and elastomeric proteins in general where β-turn-related structures serve as fundamental units of the structure and elasticity.  Meanwhile, the high-hydrophobicity sequence of the Exon III domain is proposed to play a critical role in resilin energy storage and transduction.

a) Experimental approach to measure expansion-contraction transition of resilin molecules using the Single Molecule Force Spectroscopy method through AFM. (b)  Correlated computational free energy profile for the expansion-contraction transition of resilin. PMF profiles along the reaction coordinate of the radius of gyration will be obtained from umbrella sampling with weighted histogram analysis method (WHAM).

Professors Timothy Vaden and Gregory Caputo, Dept. of Chemistry and Biochemistry, Rowan University

Project Background: Ionic liquids (ILs) have recently found a variety of novel biomedical and biochemical applications. ILs have been used to solubilize biomaterials, inhibit or enhance enzyme activities, enhance antibiotic effectiveness or act as novel antimicrobial compounds, act as drug delivery systems, and even function as cosmetics formulations ingredients. A natural question arises when studying ILs and biological systems regarding the nature of interactions between ILs and biomolecules. This question is central to the development of safe, useful, and effective ILs for biomedical applications. Hence, numerous recent investigations have reported protein – IL interactions and protein structures and stabilities in the presence of ILs in aqueous solution, and have provided models of protein interactions with ILs from molecular dynamics simulations and spectroscopic characterization. Most studies have focused on simple helical proteins such as BSA, lysozyme, myoglobin, etc. in isolated aqueous solutions, and have focused on equilibrium experimental conditions. We are interested in understanding how ILs interact with proteins different of secondary structure motifs (in β-sheet, β-barrel, and other complex structures), in membranes or other volume-restricted physiological environments, and how the ILs affect protein dynamics.

Project Description: The project will combine the biophysical chemistry experience of three groups in the Dept of Chemistry and Biochemistry at Rowan University in order to understand the molecular interactions between ILs and -sheet, -barrel, and membrane proteins, how these interactions affect the protein conformational dynamics, and how these effects differ between proteins in dilute buffer solutions and in membranes or confined environments. Dr. Gregory Caputo (biochemist) will synthesize and purify proteins from genetically coded E. Coli or other bacterial sources. Dr. Timothy Vaden (biophysical chemist) will prepare ILs, prepare proteins in various samples and environments, and characterize proteins using IR spectroscopy, CD spectroscopy, and mass spectrometry. Dr. Vaden will also perform thermodynamics (protein unfolding) and kinetics/dynamics measurements using fluorescence correlation spectroscopy (equipment at a different university). Dr. Chun Wu (computational chemist) will run molecular dynamics simulations to probe the specific IL-protein interactions, and quantify the atomic and molecular-level pictures in terms of protein structures, stabilities, and dynamics. Most equipment is in place and the budget will provide for faculty summer salaries, student salaries, consumable materials and equipment (test tubes, etc), and some minor spectroscopy equipment (eg, a time-resolved fluorimeter).

Project Aims and Goals: The project aims to use experiments and simulations to understand the effects of ILs in solution on various proteins of “real-world” applicability in different environments. The goals are (1) to develop an understanding of how ILs affect protein structures and conformational stabilities; (2) to develop novel ILs that would be biocompatible and have “designed” effects of proteins in physiologically-relevant environments; and (3) to quantify the differences between protein-IL interactions in solution and protein-IL interactions in cell membranes.

 Broader Impacts: Rowan University (especially the Dept. of Chemistry and Biochemistry) is a primarily undergraduate research institution and the project will train undergraduate students. We also hope to develop the investigations into laboratory experiences for our upper-level physical chemistry, biophysical chemistry, and molecular modeling courses. These experiences would demonstrate the power of combining molecular simulations with biochemistry and spectroscopy experiments to understand the interactions involving a protein at the molecular level for “novel” systems.

Publication: Borrell, K. L.; Cancglin, C.; Stinger, B. L.; DeFrates, K. G.; Caputo, G. A.; Wu, C.*; Vaden, T. D., An Experimental and Molecular Dynamics Study of Red Fluorescent Protein mCherry in Novel Aqueous Amino Acid Ionic Liquids. The Journal of Physical Chemistry B 2017, 121 (18), 4823-4832.

Professor Dimitri Pestov, Department of Cell Biology, Rowan University-School of Osteopathic Medicine

Targeting ribosome biogenesis as a new strategy to enhance cancer chemotherapy.

Chemotherapy remains the mainstay of cancer treatment, but it is invariably associated with serious side effects. In addition, cancer cells often develop resistance to chemotherapeutic drugs, leading to treatment failure. Thus, new approaches to chemotherapy are needed for improving clinical outlook and quality of life in cancer patients. The goal of the proposed study is to develop a new class of pharmacological agents that will make chemotherapy safer and more effective. Our recent studies identified cellular proteins involved in the assembly of ribosomes as a promising molecular target for making normal cells in the body more resistant to anticancer drugs. We found that by temporarily disabling activities of one of these proteins called Bop1, we could protect cultured cells from treatments with high doses of chemotherapeutic drugs camptothecin and metothrexate that would normally be lethal for these cells. Furthermore, this protection requires the controls genome stability in normal cells but is often lost in human cancers. Because cytoprotection through Bop1 only works in noncancerous, p53-positive cells, resistance of normal cells can be selectively increased while targeting p53-negative cancer cells with chemotherapeutic drugs. To start the development of novel pharmacological agents based on these findings, we will first use advanced computational tools to pinpoint parts of the Bop1 molecule that could be targeted via small-molecule chemical compounds. The predicted druggable surfaces in Bop1 will be validated by introducing point mutations into Bop1 and biochemical assays. Next, we will virtually dock millions tens of thousands of small molecules to the selected parts of the Bop1 structure to identify candidate inhibitors. By using in silico drug discovery at the initial stages, we expect to dramatically reduce the time and costs necessary to identify chemical compounds that will inhibit Bop1 function. In the future research, these pharmacological candidates will be used to test cytoprotection-enhanced anticancer drug treatments in animal models, moving us a step closer to improved chemotherapeutic interventions in human cancers.

Homomolgy model of mouse Wdr12-Bop1 (purple-blue) and some key Bop1 residues for mutations.

Professors Moura-Letts Gustavo and Thomas M. Keck, Chemistry & Biochemistry, Rowan Univ.

Understanding the Consequences of 6TM Splice Variants in MOR-1 Ligand Binding: Towards Novel Analgesics

Opioid drugs are a critical class of medications to treat acute and chronic pain, a serious and costly public health issue. The prototypical opioid analgesic morphine (Figure 1) is on the WHO Model List of Essential Medicines, the most important medications needed in a basic health system.  However, medical use of opioid analgesics is limited by critical side effects including sedation, constipation, abuse liability and respiratory depression. There is pressing need for the development of novel opioid analgesics with more precisely targeted mechanisms of action. Opioid analgesics induce their therapeutic effects primarily through the activation of the μ opioid receptor (MOR-1), found on a wide variety of central and peripheral neurons. MOR-1 is a seven transmembrane (7TM) G protein-coupled receptor (GPCR). Many splice variants of the MOR-1 gene, OPMR1, in rodents and humans are actively expressed and appear to physiologically mediate analgesic responses. Notably, a subset of truncated variants of OPMR1 have altered sensitivity to morphine and other classical opioid analgesics in vitro and are predicted to contain only six TM domains (6TM). Recent studies have identified the novel analgesic IBNtxA (Figure 1) as a preferential agonist of 6TM variants, providing potent analgesia with reduced side effects compared to morphine. These results raise the hope that it might be possible to dissociate opioid analgesia from side effects and abuse liability. Thus, targeting 6TM variants may yield important new analgesics in the future. However, there is no high-resolution receptor/complex structure of the 6TM MOR variants, and it is not clear how the receptor truncation alters the ligand binding properties of these receptors. Although IBNtxA is a promising drug candidate, its high molecular complexity derived from its substituted oxymorphone alkaloid core may prevent it from further development as a clinical drug. Our overall goal is to develop novel ligands selective toward the human MOR-1 6TM splice variants (hMOR-1G1 and hMOR-1G2) using combined computational and experimental approaches. Based on preliminary docking data, two simplified scaffolds of IBNtxA have been identified as initial lead structures. However, the number of possible ligands is prohibitively large for synthesis and in vitro screening. We will apply our computational screening to reduce the number to ~50, which will then synthesized and tested in vitro.

                                   Figure 1 Chemical structures of morphine, IBNtxA and proposed novel scaffolds.