Characterization of RAS Protein Using Raman Spectroscopy

Abstract

Ras is a family of related proteins expressed in all animal cells, which is small GTPase anchors to the plasma membrane where it regulates signal transduction for cell proliferation, growth, survival, and other cell functions. Mutation of Ras makes it lost the ability to regulate signal transduction and causes cancer. Three different types of Ras (KRAS, HRAS, NRAS) and their isoforms have been identified in different human cancers. Understanding Ras protein functioning mechanisms is crucial in developing cancer drug and cancer therapy at the molecular level, which is highly related to its primary, secondary, and tertiary structures. At present, X-ray crystallography and NMR are the two major techniques to effectively measure Ras protein structures and its conformational geometries, but they cannot measure Ras protein conformations in cells under physiological conditions. In this study, we intend to develop label-free Raman spectroscopy methods to characterize and quantitate Ras isoforms and their structural conformations with their unique finger-printing vibrational spectra in vivo and in vitro, as well as to monitor the small drug molecule’s binding with Ras molecule in live cells, which is new to Ras based cancer biology. The first part of this dissertation work is to explore the capability of near-infrared Raman spectroscopy and multivariate analysis techniques for the characterization and differentiation of Ras isoforms and their structural conformations with their finger-printing Raman spectra. We have collected Raman spectra from different Ras isoforms. We have developed a principal component analysis (PCA), a discriminant analysis of principal component (DAPC), and Raman barcode methods, and demonstrated that Ras isoforms can be differentiated by their Raman spectra. We have studied the structural conformations of Ras proteins with GDP and GTP loading. By deconvolution of Raman spectra, we were able to obtain new information about Ras conformational structures including protein’s secondary structures, hydrogen bonding condition of phenol side chain, and hydrophobic nature of tyrosine doublet for each Ras isoforms. We have applied Raman spectroscopy for the study of the specific inhibitor (ARS1620 drug) and KRAS G12C interactions, offering new insights into Ras-targeted cancer therapies. These data allow generating a library of Raman spectra of Ras isoforms in vitro, which may serve as the control and platform for developing methods to detect the Ras Raman fingerprint inside a cell. The second part is to develop a vacuum-enhanced micro-Raman spectroscopy (VERS) for the detection of analyte molecules at relatively low concentrations in an aqueous medium. Although Raman spectroscopy is a powerful technique for analyzing biomolecules, the molecular cross-section of Raman scattering is very low, and thus, high analyte concentration in hundreds of mM is typically required for normal Raman spectroscopy. Since Ras proteins are generally available at low concentration either in vitro or inside the live cells, we intend to develop a novel label-free enhanced Raman spectroscopy for the detection of proteins and other molecules at relatively low concentrations. The VERS technique relies on the increase in the molecule’s concentration within the micron-sized excitation volume of laser focus by vacuum evaporation of solvents and does not cause the alteration in normal Raman spectra. We demonstrate that an enhancement factor of ~103-104 was observed for glucose and protein samples, and the enhancement factor depends on the size of the sample holder and the volume of the liquid sample used. We show that VERS can be used to detect ciprofloxacin antibiotics in human urine at a level of 2 μg/ml. We also show that the VERS technique is particularly useful for detecting biomolecules resolved in a solvent such as dimethyl sulfoxide (DMSO) that has an intense Raman background due to the evaporation of the solvent. Traditional enhancer materials like metal nanoparticles are not needed in VERS technique. The third part is to develop the surface-enhanced Raman scattering (SERS) spectroscopy and SERS-based super-resolution Raman imaging techniques for the detection and chemical imaging of analytes at the single-molecule level. Since the intrinsic Raman scattering of Ras protein and other biomolecules has very low probability and the intra-cellular concentration of Ras is extremely low (in nanomole level), we intend to introduce nano-sized metallic particles (SERS substrate) to bind with the target molecules and use their SERS signals to detect the target molecules with an ultrahigh sensitivity and precisely determine the spatial location of the molecules. We built a home-made experimental setup that allows simultaneous observation of the Raman images of hot spots and acquisition of time-lapse Raman spectra of a single hot particle. We explored the dynamic behavior of SERS of hot particles at the single-molecule level, focusing on the influence of environmental conditions and laser power on the stability of SERS Raman signals. It was observed that SERS intensity fluctuations were more pronounced at the atmospheric pressure than at a low pressure. The experimental data further revealed that as the laser power increased (~ 8.9 μW and above), there was a notable increase in SERS signal fluctuations and blinking, probably due to thermal or photo-induced changes in nanoparticle clusters. We proved that the use of bright field microscopy and Raman imaging for tracking hotspots together with ThunderSTORM software, was effective in accurately identifying and analyzing the change in hot particle’s center position. These findings underscore the importance of precise experimental control on both laser parameters and environmental conditions to optimize SERS applications at single molecule levels, which advances our understanding of SERS at the nanoscale, promoting its application in nanotechnology and materials science.