Biomolecular Spectroscopy

ABOUT THE COURSE:This course will cover the established spectroscopic techniques for the study of biomolecules, along with their practical applications. This course will assist students in selecting and assessing appropriate spectroscopic techniques for studying biomolecules.INTENDED AUDIENCE: M. Sc./ PhD, Teachers and Scientists working in academic institutes and industriesPREREQUISITES: Under graduate level understanding of Chemistry & BiologyINDUSTRY SUPPORT: Biocon, Wockhardt, Aurobindo Biopharma etc.

Syllabus

Week 1: Absorption Spectroscopy: Theory of absorption of light by molecules: propagation of an electromagnetic wave through space. Jablonski diagram. Instrumentation. Beer’s law and the extinction coefficient. Chromophore: factors affecting the absorption properties of a chromophore. solvent-induced heterogeneity, red-shifts and blue shifts; polarity vs polarizability of the medium
Week 2:Absorption Spectroscopy (contd). Spectral shifts in rhodopsin and vision. Structural studies DNA using absorption spectroscopy. Helix coil transition of double stranded DNA. Melting curve of DNA: Hyperchromicity & hypochromicity. Structural studies of proteins using absorption spectroscopy. Practical Considerations: Spectral bandwidth, wavelength error & stray light
Week 3:Circular Dichroism (CD) Spectroscopy. Introduction. Linearly and circularly polarized light. Instrumentation. Experimental: sample concentration and cell pathlength. Solvent effects. Characteristics of CD & ORD (optical rotatory dispersion) spectroscopy: Cotton Effect. Identification of known optical isomers
Week 4:Circular Dichroism (CD) Spectroscopy (contd). Determination of protein secondary structure. Detection of molten globule like structure of proteins. Determination of nucleic acid conformations. Use of CD spectroscopy to study stacking orientation of the base pairs. Applications of CD spectroscopy in studying amyloid based neurodegenerative disorders
Week 5:Fluorescence Spectroscopy. Theory of fluorescence spectroscopy. Photoluminescence, emission, and absorption. Stokes Shift. How does a fluorescence spectrophotometer work? Solvent and Environmental Effects on Fluorescence. Inner filter effect in fluorescence.
Week 6:Fluorescence Spectroscopy (cont.). Intrinsic fluorescence measurements for studying biomolecules. Protein fluorescence. Extrinsic fluorescence. Fluorescence resonance energy transfer. Fluorescence quenching
Week 7:Fluorescence Spectroscopy (cont.). Fluorescence quenching (contd.). Stern-Volmer Equation. Fluorescence Anisotropy: Brownian motion and photo selection. Fluorescence Anisotropy (contd.). GFP and its applications. Novel Fluorophores: Quantum Dots. Time-resolved Fluorescence and its applications for protein dynamics
Week 8:Infrared (IR) Spectroscopy. Theory of Infrared absorption. Introducing FT-IR: Interferometry. Instrumentation: IR sources, detectors, and beam splitter. FTIR analysis of proteins: amide I and amide II bonds. Studying DNA/RNA conformational changes.