2016

Undergraduate Research Conference

Meet the 2016 URC Presenters from Cheng Lab

By Tansy A. Gu
Real Time Tracking of Protein Synthesis and Assembly With Genetically Encoded Small Illuminant (GESI)

There are many probes and sensors used in molecular biology to track cellular activity such as the green fluorescent protein (GFP). Compared to the 238 amino acid long GFP, the genetically encoded small illuminant (GESI) is only 12 amino acids. GESI acts as a fluorogen activating protein (FAP) that can bind the dye malachite green (MG) to produce a fluorescent signal when excited by light of near infrared wavelength. GESI was cloned at the C terminus of hepatitis E virus (HEV) capsid protein gene and chimeric VLPs were produced in insect cells. The HEVGESI VLP was used to visualize protein localization in cells and quantify protein content in vitro. MG binding to GESI was found to be highly specific as seen by immunostaining with capsid antibodies. Fluorescence intensity increased overtime when GESI VLP was incubated with MG on a Native-PAGE gel. GESI fluorescence is a good indicator of protein content as it correlated with absorbance 280. GESI was used to track viral production and protein synthesis in insect cells on different days post infection by subcellular fractionation. Using the GESI VLP, we plan to study protein folding and virus assembly in real time through fluorescence correlation spectroscopy
By Juliana W. Noronha
Conjugation of Genetically Engineered HEV-VLPs to Gold Nano-Clusters (Au102(pMBA)44) for VLP Tracking in Cells

The capsid structure of Hepatitis E Virus (HEV) was modified to produce non-infectious virus-like particles (HEV-VLPs). VLPs retain the antigenicity, structural stability and cell binding capacity of the native virus. An Aspargine residue on the surface of the VLP was mutated to Cysteine (N573C), allowing the particle to covalently bind and expose ligands. This modulatable HEV capsid has potential to become a stable antigen and/or drug delivery platform. To study the capacity and efficiency of this platform, tracking VLPs in cells is critical. While purified virus-sized proteins are visible in cryo-electron microscopy (cryo-EM), distinguishing virus sized particles in cells remains a challenge due to labelling limitations. This study investigates conjugation of gold nano-clusters, Au102(pMBA)44, to the HEVVLP cysteine via ligand exchange. Au102(pMBA)44 particles are ligand protected mono-dispersed nano-gold clusters with high electron density, allowing VLP structure detection and tracking in cryo-EM. For conjugation optimization, incubation time, temperature and reducing agents were compared. Following Au102(pMBA)44 conjugation to HEV-VLP, the focus turns to cryo-EM and 3D reconstruction to assess the degree of Au102(pMBA)44 occupancy on the VLP cysteine sites.
By Aria Sikaroudi
Progressive Improvement of 3D Structure Resolution of HEV VLP Bound to Functionalized Gold-Nano Clusters (Au102-C6MI)

Virus-like particles (VLPs) have been used as biologically stable nanocarriers for targeted delivery of immunogens and/ or small molecules for vaccines and therapeutics. The Hepatitis E Virus (HEV) capsid protein was genetically engineered to form a recombinant, self-assembling VLP with exposed surface cysteines for covalent conjugation. Water-soluble nano-gold clusters (Au102(pMBA)44) containing a functional group, six carbon-maleimide linker (C6-MI), were covalently bound to the VLP cysteines. Resolving the structural features of HEV-VLPs upon Au102-C6 MI conjugation is a crucial step in understanding the architecture of this novel drug-delivery platform. It is also important for determination of structural differences in the HEV-VLP upon Au102-C6MI conjugation, in locating the stable position of Au102-C6MI on the VLP surface, and in determining whether gold nanoclusters can improve the resolution of 3D structures via cryo-Electron Microscopy and Single Particle Reconstruction (SPR) methods. A preliminary 3D structure of HEV-VLP conjugated with gold linker arms on the cysteines was generated. In order to improve the resolving power of structural features, the 3D structure was subjected to progressive resolution thresholding and refined through a systematic classification of highly representative imaged particles.
By Ryan B. Ko
Determination of Metastable Conformations of HIV-1 Env Thorough Single Particle Reconstruction and a Statistical Population Isolation Approach

The envelope protein (Env) is a main target for neutralizing antibodies against human immunodeficiency virus (HIV- 1). Env consists of 3 units of gp120 and gp41, assembled in a trimeric array. Transmission electron microscopy (TEM) and Single Particle Reconstruction methods were used to generate preliminary 3D density maps of Env in its native state. Based on reference-free class averages (RFCA), a gradient of open and closed conformations was observed; suggesting the existence of particle heterogeneity and metastability of native Env conformations. A statistical analysis approach was utilized to segregate populations of open and closed conformations from the heterogeneous mixture. Sub-populations were then grouped together by orientation and based on calculated Quality Factor Scores (QFS), which are dictated by the cross-correlation of individual imaged particles and their relative 2D re-projections. The subpopulations were then utilized to generate high resolution 3D representations of Env in its open and closed conformations, which are critical for subsequent studies of neutralizing epitope exposure or occlusion. Our statistical analysis approach is also critical for reconstructing high-resolution 3D structures of Env, and can be used as a tool to measure the degree of heterogeneity within a dataset.