Biophysics II

Plan of the lecture:

1. Relaxation methods in molecular biophysics: theoretical basis of relaxation methods, experimental methods, T-jump, Electric field-jump, stopped-flow.
2. pH dependence of molecular structure and function.
3. Investigation of protein folding and structural transitions in nucleic acids by stopped-flow and T-jump methods.
4. Application of electric field jump methods in studies of long-range structure of large bimolecules.
5. Application of Brownian dynamics, molecular dynamics and molecular electrostatics methods in studies of proteins and nucleic acids.
6. Protein crystallography - principles and possibilities of the X-ray structure analysis of proteins, physical and theoretical background, experimental techniques, steps in the X-ray analysis of crystalline proteins, problems associated with every step, phase problem, resolution.
7. Crystallization of proteins - factors affecting solubility of proteins, and crystal nucleation and growth, phase diagram for protein solubility, commonly used precipitants, techniques of crystallization - methods of hanging and sitting drop, dialysis, micro and macroseeding, screens; quality and properties of protein crystals.
8. Sources and detectors of X-rays, methods for solving phase problem and obtaining protein structures - Patterson function, molecular replacement, isomorphous replacement, anomalous scattering; preparation of heavy atom derivatives, selenomethionine; data collection, cryocrystallography; refinement, structure quality; time-resolved X-ray analysis, Laue diffraction.
9. General characterization of nuclear magnetic resonance techniques in light of its application to biology
10. Studies of metabolism of intact cells and tissues by in vivo NMR
11. Magnetic resonance imaging (MRI)
    1. Cancer diagnostic
    2. Angiography
    3. NMR microscopy
    4. Functional NMR (fNMR)
12. Merging of the in vivo NMR and MRI techniques: topical magnetic resonance (TMR) and chemical shift imaging (CSI)
13. Dynamics and structures of biological membranes by means of broad line and cross-polarization magic angle spinning (CPMAS)
14. Basic knowledge on fluorescence and phosphorescence emission of molecules - Jablonski diagram; differences between fluorescence and phosphorescence; Stokes shift; mirror image rule.
15. Instrumentation for emission spectroscopy - typical spectrofluorimeter and phosphorimeter; quantum yield; emission and excitation spectra; examples of (i) fluorescence and phosphorescence spectra of tyrosine, tryptophan and phenylalanine, and (ii) tryptophan protein (deoxycytidine kinase).
16. Measurements of lifetime of the excited states - time-correlated single photon counting (TCSPC) method; fluorescence and phosphorescence intensity decays.
17. Measurements of lifetime of the excited states - detection of phase and modulation; comparison with TCSPC.
18. Interpretation of fluorescence and phosphorescence intensity decays.
19. Simultaneous absorption of two photons - experimental confirmation of theoretical predictions.
20. Similarity and differences between one-photon (OPIF) and two-photon induced fluorescence (TPIF) - examples of (i) one-photon excitation spectra (OPE) and two-photon excitation spectra (TPE) of tyrosine, tryptophan and phenylalanine, (ii) effect of excitation of the fluorescence emission spectra of tyrosine and tryptophan in solution and in proteins.
21. Polarisation (anisotropy) of excitation and emission - measurements in liquid phase, and in low-temperature glasses; analysis of absorption transitions in tryptophan using excitation anisotropy spectra.
22. Effect of multi-photon excitation on the limiting anisotropy values - photoselection rules; examples of (i) anisotropy of tyrosine and tryptophan fluorescence in solution and in proteins, (ii) low (~ 0) anisotropy of tyrosine fluorescence resulted from TPE, and (iii) high TPIF anisotropy of reduced form of nicotinamide-adenosine dinucleotide (NADH) in liquid phase, glassy solutions, and in the complexes with horse liver alcohol dehydrogenase (LADH).
23. Fluorescence anisotropy decays - rotational correlation time and its relation to molecular dynamics.
24. Multi-photon techniques in confocal microscopy - comparison of OPIF and TPIF.
25. Structural images of the cell and tissues - fluorescence probes.
26. Circular dichroism (CD) spectroscopy, and its application in protein structure studies.
27. Infrared (IR) absorption spectroscopy - examples of (i) identification of dynamic tautomeric equilibria of the promutagenic analogues of nucleic acid bases, (ii) effect of base-pairing (hydrogen bonding) with potentially complementary bases on the tautomeric equilibria, and (iii) explanation of the molecular mechanism of transition mutations.