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Welcome to 2011 UK CD and LD winter workshop

Welcome to the 2011 UK CD and LD Winter Workshop! Join us as we explore the principles and applications of Circular Dichroism spectroscopy in chemistry and biology. Learn about the absorption of circularly polarized light by chiral molecules and how it can be used to analyze protein structures. Safety guidelines and workshop schedule included.

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Welcome to 2011 UK CD and LD winter workshop

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  1. Welcome to 2011 UK CD and LD winter workshop • Thanks to MOAC, Department of Chemistry, and the University of Warwick for hosting us.

  2. Safety • In the lab: lab coats, safety spectacles, gloves as appropriate. • In the event of MOAC fire alarm go outside the front door. • In event of chemistry fire alarm – we meet outside library. No lifts! • Elsewhere read signs.

  3. Where will we be?? • Lectures and computer workshops: • MOAC EPSRC Doctoral Training Centre • Laboratory work: • Mainly chemistry • Food: • Lunches: DIY – including clear-up! Use the time to get to know other people. • Coffee/Tea/Squash: available – again DIY. • Dishwasher need stacking with dirty dishes and emptying when clean! • Tuesday dinner with, posters etc. in MOAC. • Wednesday: Browns in centre of the city

  4. More things • Keep the common room tidy • READ but not REMOVE books! • Internet access via the common room computers. Ask about wireless if you have a laptop. • Wear your name badges. • Receipts/refunds on deposit see me of you haven’t got one.

  5. People • All organisational credit to: • Anne Maynard and Fiona Friel • Big problems: Alison Rodger • Warwick students • Lecturers and course leaders – see programme.

  6. Circular Dichroism Spectroscopy Introduction Alison Rodger, University of Warwick a.rodger@warwick.ac.uk

  7. Absorbance Bonds  Molecules  Spectroscopy Molecules are 'glued together' by electrons between the atoms. ~ Two electrons per bond. Two types of bonds are important for bio molecules: s (sigma) bonds look like s-orbitals when viewed along the bond axis.  (pi) bonds look like p-orbitals (dumbbells) when viewed down the bond axis Also non-bonding pairs, e.g. lone pairs on N & O Ultra violet/visible (UV/vis): how much energy is required to push electrons to new orbitals.

  8. Electrons  bonds  structure UV/visible light:l ~ 180 nm – 800 nm, energy hn = hc/l causes electrons to go to higher energy levels. l required depends on electron rearrangement needed. In solution: broad bands due to diff. vibrational levels in excited state & molecules having slightly different energy levels. E UV: –350 nm Vis: 400 nm – Excited electronic state • . Ground electronic state Ground vibn’l level r

  9. Absorption spectra of DNA & proteins • With proteins and DNA the transitions we usually study • are n * and * so can access them with • normal spectrometers. • Below 200 nm need nitrogen purging because O2 absorbs • Absorbance: A = log(Io/I) • = log(intensities in/out) • Beer Lambert A = ecl, e extinction coefficient (units?), • c concentration, l length (cm)

  10. view looking down the beam polarizer Before polarization: unpolarized beam After polarization: linear polarized beam view looking down the beam Linearly polarized light • All photons in a beam of light have an electric field vector, E, that is at right angles to the direction of travel of the beam, x, and varies as a sine wave x E E E E

  11. Circularly polarized light • Add two linearly polarized light beams - both propagating along x • y-polarized + • z-polarisedbut starting ¼ wavelength out of phase from the y one. Ey= msin(2py/l) Ez= mcos(2pz/l) The two waves add together to form right handed (clockwise) circularly polarized light. y y view looking down the beam x or time z z

  12. Circular Dichroism • CD is the difference between the absorption of left and right handed circularly polarized light as a function of wavelength. • The difference very small (~<<1/1000 of total) • DA(l) = AL(l)-AR(l) = [eL(l) - eR(l)]lc or • DA(l) = De (l)lc • De ~ typically < 10 M-1cm-1 vs. e~20,000M-1cm-1 • CD is very small difference between two large signals A CD

  13. Circularly polarized light Linearly polarized light: Electric vector direction constant—magnitude varies. Circular polarized light: Electric vector direction varies—magnitude constant

  14. CD Spectra Varying absorption of circularly polarised light by chiral molecules results in distinctive spectra under absorption bands Need chiral light and chiral molecule to get CD spectrum of a solution

  15. CD spectropolarimeter Max light intensity: 300-400 nm Xenon lamp Quartz 1/4l- plate. Oscillates at ~ 50 Hz  CPL M = mirror P=prism Monochromater prism CD=A/(absorbance units)=4(degrees)/(180ln10) CD=(millidegrees)/32,980

  16. Empirical analysis of CD HPLC detector Structural change

  17. CD to answer: does it change structure? Reduced and unreduced plant defensins

  18. a-helix 20 15 b-sheet 10 turn 5 0 -5 -10 Poly proline type II 180 200 220 240 260 Wavelength/nm The CD signal for a protein depends on its secondary structure • Two regions • Secondary structure (170 – 260 nm) • Aromatic region (240 – 360 nm) • Secondary structure • - Amide backbone • Aromatic • - Phenylalanine (250 – 270 nm) • -Tryptophan (260 – 300 nm) • - Tyrosine (270 – 290 nm) • - Disulfide bonds • a-helical protein spectra are • distinctive: • 222,208,~190 nm

  19. Amino acids, peptides and proteins H H

  20. CD  secondary structure of protein • Fit the unknown CD curve qu to a combination of standard curves • In the simplest case use the standard spectra for secondary structures • qt = xaqa + xbqb + xcqc • Vary xa, xb and xc to give the best fit of qt to qu • while xa+ xb + xc = 1.0 CD spectra can be analysed by the structure-fitting program “cdsstr” (of C.J. Johnson) to obtain % of secondary structure motifs. Cdsstr uses a basis set of protein spectra • fits best with: • xa= 80%; xb=0%; xc= 20% • agrees well with structure: • 78% helix,22% other

  21. CD signals are sensitive to secondary structure: coiled-coil (2 a-helices twisted) Salt bridge hydrophobic Characteristic heptad repeat abcdefgabcdefgabcdefgabcdefg MKQLEDKVEELLSKNYHLENEVARLKKL

  22. The CD signal for a protein depends on its secondary structure —— chymotrypsin (~0.1 helix, 0.15 b-sheet, 0.15 b-turn) —— lysozyme (mixed 0.4 helix, 0.2 turn) —— triosephosphate isomerase (mostly a some b) —— myoglobin (all a)

  23. Hydrophobic region Transmembrane -helix SPP TPP N C Protein conformation as a function of environment Tris buffer soluble Pre-PSbW and and Unfolded using Guanidinium Chloride Pre-PsbW, thylakoid membrane protein ‘No structure’ in guanidinium chloride Some in tris buffer (multimer) Lots in SDS micelles (folded, 2 helices)

  24. -  - - CDsstr results for pre-PsbW

  25. Lots of a-helix 1.4 mg/mL; 0.01 cm; 42% α-helix 208 nm, 100% a-helix = 12 mol-1dm3cm-1 Antibody Other helix = 9% PPII 0% α-helix 10% other helix 33% β-sheet 14% turn 42% other

  26. Largely b-sheet protein 0.01 cm; 0.3 mg/mL; 16% PPII 19% β-sheet 15% turns 47% other Typical mixed a-b-sheet protein spectrum 15% α-helix 11% other helix 26% β-sheet 12% turns 36% other

  27. Near UV: protein CD

  28. CD requires helical electron motion Require magnetic dipole transition moment  0 Require electric dipole transition moment  0

  29. CD from coupled oscillators R = CD strength = Im(.m) =electric dipole transition moment m=magnetic dipole transition moment -* transition of a helical polypeptide High energy, 190 nm Low energy, 208 nm

  30. Oriented CD spectra

  31. Vancomycin & ristocetin Glycopeptide antibiotics that prevent cross-linking and transglycosylation during bacterial cell wall formation. Noncovalent dimerisation plays a key role in their activity CD used to give binding constants V-V, V-R and V-peptides, R-peptides. Assume non-covalent dimers. CD change (induced CD)  [dimer] Kdimerisation= 205 (mM)-1 V + R V-R

  32. Nucleic acid CD DNA and RNA polymers:sugar units of the backbone provide the chirality, but not the chromophores. CD spectrum of a polynucleotide arises from interaction between the * transitions of stacked bases. But note isolated nucleotides are chiral Use CD to identify which polymorph: CD varies more with base orientation than sequence

  33. Nucleic acid CD spectra Calf thymus DNA: B-DNA (10.4 bases) A-DNA B-DNA (10.2 bases) B-DNA: 72%, 50% & 31% GC content Poly[d(G-C)] 2: B-DNA A-DNA Z-DNA B-DNA: 275>0, 258=0, 240<0, 220>/=0, 180/190>>>0 A:DNA: 295</=0, 260>>0, 250230>/=0, 210<<0, 190>>0 Z-DNA: 290<0, 260>0, 195/200<<<0, 185 180=0

  34. RNA: CNG repeats (neurological disorders e.g. Myotonic Dystrophy ) Which is melted? Unusual RNAs: adopt duplex A-form plus something else. ??? Triplex.

  35. [Ru(phen)3]2+ ct+ high r  ct+ low r AT GC

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