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Foundations of Structural Biology I

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Foundations of Structural Biology I Foundations of Structural Biology II: GMS BY 763 Computation, Nuclear Magnetic Resonance, Thermodynamics and Spectroscopy in Structural Biology Spring Semester 2015: Monday 1:00 – 3:00 pm Course Director: Dr. Atkinson, W302, 8-4015 5 January Dr. Atkinson Introduction, Structural Databases and Tools: Protein Data Bank (PDB), PDB structure files, EM Structure repository (EMDataBank) 12 January Dr. Atkinson Structural Computational Biology I Macromolecular graphics, structure visualization and analysis. 19 January Martin Luther King Day – no class 26 January Dr. Atkinson Structural Computational Biology II Molecular Mechanics and Dynamics 2 February Dr. Gursky Thermodynamic Methods I Protein Energetics - what is it good for? Protein folding, stability and alternative conformations. Free energy landscape. Thermodynamic and kinetic stability; Gibbs and Eyring equations. Statistical weight and probability; extensive &intensive variables, state functions. Three laws of thermodynamics. Entropy S in spontaneous and equilibrium processes 9 February Dr. Gursky Thermodynamic Methods II A Long Way to Gibbs-Helmholtz Equation: Le Chatelier’s principle. Internal energy E and enthalpy H. Heat capacity - microscopic meaning, typical values. Effect of apolar compounds on Cp of liquid water. Protein unfolding as a 1st order phase transition. Gibbs free energy G and its linkage to H and S. Typical values of Delta S, Delta H and DeltaG for globular proteins. Entropy-enthalpy compensation in globular proteins. 16 February President’s Day – no class 23 February Dr. Gursky Thermodynamic Methods III Cool and Hot Methods in Protein Thermodynamics Gibbs-Helmholtz equation and its application to proteins. Cold denaturation. Plot G(T) and geometrical interpretation of Tm, Tc, S, H, and Cp Differential scanning calorimetry (DSC) – principle, advantages and limitations. Van’t Hoff and calorimetric enthalpy. Application to single- and multidomain proteins and protein folding intermediates. Spectroscopic methods for the analysis of protein thermal stability. Melting data and Van’t Hoff analysis in thermodynamics. Temperature-jumps and Arrhenius analysis in kinetics. 2 March Dr. Gursky Spectroscopic Methods I Spectroscopic Methods of Protein Thermodynamic and Structural Analysis: Comparison of the calorimetric and spectroscopic approaches to protein thermal denaturation. Chemical denaturation: spectroscopic analysis of thermodynamic and kinetic stability. Protein stability probed by mutagenesis. 9 March Spring Recess – no class 16 March Dr. Gursky Spectroscopic Methods II Infrared and Raman spectroscopy for structural and chemical analysis of macromolecules. Molecular vibration, stretching and bending modes. Principal IR bands for the peptide group as a function of secondary structure. Fourier Transform IR spectrometry (FTIR) for secondary structural analysis. Raman spectroscopy; resonance Rama and bioimaging methods. Review of light polarization and light absorption; Beer’s law; normal absorption, linear and circular dichroism. Far-UV CD spectra of pure secondary structures. Near-UV CD and tertiary protein structure. Spectral deconvolution; quantitative secondary structural analysis of proteins using CD and FTIR. 23 March Dr. Gursky Spectroscopic Methods III Fluorescence Spectroscopy for protein structural and folding studies: Relation between absorption and emission spectra. Factors affecting fluorescent intensity. Steady state and kinetic measurements. Intrinsic and extrinsic fluorophores. Fluorescent quenching as a probe for solvent accessibility of protein chromophores. Probing folding states by fluorescent dyes. Forster resonance energy transfer for distance measurements. 30 March Dr. McKnight Structural NMR I Introduction to Fundamental Aspects of NMR: Nuclear spin, Zeeman splitting, Boltzman distribution, precession of spins, Bloch equations, one pulse NMR experiment, spin relaxation, linewidth, chemical shifts, j-coupling, dipole-dipole interactions (NOE). 6 April Dr. McKnight Structural NMR II Experimental Aspects of NMR: Sample considerations and conditions. Instrumentation: Tour of an NMR spectrometer, data acquisition, sampling theorem, quadrature detection, phasing, lock channel. Data processing: Fourier transforms, apodization, zerofilling, linear prediction, referencing, and integration. Water suppression: Presaturation, gradient, jump-return, spinlocks, solvent deconvolution. 13 April Dr. McKnight Structural NMR III Multidimensional and Heteronuclear NMR: Through bond experiments: COSY and TOCSY. Through space experiments: NOESY and ROESY. Heteronuclear experiments: HMQC, HCCH-COSY. Combining experiments: HMQC-NOESY, HMQC-TOCSY. Three dimensional experiments: 3D-HMCQ-NOESY. Triple labeling: HNCA, HN(CO)CA . Deuterium labeling of large proteins. 20 April Patriot’s Day – no class 27 April Dr. Mcknight Structural NMR IV Hydrogen Exchange and Relaxation Measurements: Hydrogen exchange rates and protections factors. Relationship of protections factors to G. Pulsed HX and protein folding. Theory and mechanisms of relaxation (T1, T2, and NOE).The spectral density function. Experimental aspects to measure T1, T2 and NOE. 4 May Dr. McKnight Structural NMR V Sequential Assignment and Structure Calculation: Proteins: Spin systems sequential NOEs, medium range NOEs, stereospecific assignments, direct methods with triple labeled samples. DNA: Spins systems, sequential NOEs. NMR distance, angle, and chemical shift restraints. Distance geometry, simulated annealing, and relaxation matrix back-calculation. Software packages. Judging the quality of NMR structures and comparison with X-ray. Spring 2015 MLK Jr. Day Monday, January 19, 2015 Classes Begin Tuesday, January 20, 2015 President’s Day Holiday, Classes Suspended Monday, February 16, 2015 Substitute Monday Schedule of Classes Tuesday, February 17, 2015 Saturday, March 7 - Spring Recess Sunday, March 15, 2015 Classes Resume Monday, March 16, 2015 Patriot’s Day Holiday, Classes Suspended Monday, April 20, 2015 Substitute Monday Schedule of Classes Wednesday, April 22, 2015 Last Day of Classes Thursday, April 30, 2015 Friday, May 1 - Study Period Monday, May 4, 2015 Final Exams Begin Tuesday, May 5, 2015 Final Exams End Saturday, May 9, 2015 Friday, May 15 - Commencement Weekend Sunday, May 17, 2015 .
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