1 Electronic SpectroscopyChem 344 final lecture topics

2 Time out—states and transitionsSpectroscopy—transitions between energy states of a molecule excited by absorption or emission of a photon hn = DE = Ei - Ef Energy levels due to interactions between parts of molecule (atoms, electrons and nucleii) as described by quantum mechanics, and are characteristic of components involved, i.e. electron distributions (orbitals), bond strengths and types plus molecular geometries and atomic masses involved

3 Spectroscopy Study of the consequences of the interaction of electromagnetic radiation (light) with molecules. Light beam characteristics - wavelength (frequency), intensity, polarization - determine types of transitions and information accessed. Intensity I ~ |E|2 l E || z B || x n = c/l x z y B | E } Polarization k || y Frequency Wavelength

4 Properties of light – probes of structureFrequency matches change in energy, type of motion E = hn, where n = c/l (in sec-1) Intensity increases the transition probability— I ~ e2 –where e is the radiation Electric Field strength Linear Polarization (absorption) aligns with direction of dipole change—(scattering to the polarizability) I ~ [dm/dQ]2 where Q is the coordinate of the motion Circular Polarization results from an interference: Im(m • m) m and m are electric and magnetic dipole Intensity (Absorbance) IR of vegetable oil n l

5 Optical Spectroscopy - Processes Monitored UV/ Fluorescence/ IR/ Raman/ Circular DichroismDiatomic Model Analytical Methods Excited State (distorted geometry) Absorption hn = Egrd - Eex UV-vis absorp. & Fluorescence. move e- (change electronic state) high freq., intense Ground State (equil. geom.) CD – circ. polarized absorption, UV or IR n0 nS Fluorescence hn = Eex - Egrd Raman –nuclei, inelastic scatter very low intensity Raman: DE = hn0-hns = hnvib IR – move nuclei low freq. & inten. Infrared: DE = hnvib Q molec. coord.

6 Optical Spectroscopy – Electronic, Example Absorption and FluorescenceEssentially a probe technique sensing changes in the local environment of fluorophores  (M-1 cm-1) Fluorescence Intensity What do you see? (typical protein) Intrinsic fluorophores eg. Trp, Tyr Change with tertiary structure, compactness Amide absorption broad, Intense, featureless, far UV ~200 nm and below

7 Circular Dichroism Most protein secondary structure studies use CDMethod is bandshape dependent. Need a different analysis Transitions fully overlap, peptide models are similar but not quantitative Length effects left out, also solvent shifts Comparison revert to libraries of proteins None are pure, all mixed

8 CD is polarized differential absorptionCircular Dichroism CD is polarized differential absorption DA = AL - AR only non-zero for chiral molecules Biopolymers are Chiral (L-amino acid, sugars, etc.) Peptide/ Protein - in uv - for amide: n-p* or p-p* in -HN-C=O- partially delocalized p-system senses structure in IR - amide centered vibrations most important Nucleic Acids – base p-p* in uv, PO2-, C=O in IR Coupled transitions between amides along chain lead to distinctive bandshapes

9 UV-vis Circular Dichroism SpectrometerSample Slits PMT PEM quartz Xe arc source This is shown to provide a comparison to VCD and ROA instruments Double prism Monochromator (inc. dispersion, dec. scatter, important in uv) JASCO–quartz prisms disperse and linearly polarize light

10 Link is mostly planar and trans, except for Xxx-ProAmino Acids - linked by Peptide bonds  coupling yields structure sensitivity Link is mostly planar and trans, except for Xxx-Pro

11 UV absorption of peptides is featureless --except aromaticsAmide p-p* and n-p* Trp – aromatic bands TrpZip peptide in water Rong Huang, unpublished

12 a-helix - common peptide secondary structure(ii+4)

13 b-sheet cross-strand H-bonding

14 Anti-parallel b-sheet (extended strands)

15 Polypeptide Circular Dichroismordered secondary structure types De l a-helix b-sheet turn Brahms et al. PNAS, 1977 poly-L-glu(a,____), poly-L-(lys-leu)(b, ), L-ala2-gly2(turn, ) Critical issue in CD structure studies is SHAPE of the De pattern

16 Large electric dipole transitions can couple over longer ranges to sense extended conformation Simplest representation is coupled oscillator Tab ma mb De = eL-eR Dipole coupling results in a derivative shaped circular dichroism l Real systems - more complex interactions - but pattern is often consistent

17 DNA B-DNA Right -hand Z-DNA Left-hand

18 B- vs. Z-DNA, major success of CDSign change in near-UV CD suggested the helix changed handedness

19 Protein Circular DichroismDA Myoglobin-high helix (_______), Immunoglobin high sheet (_______) Lysozyme, a+b (_______), Casein, “unordered” (_______), Coupling  shapes, but not isolated & modeling tough

20 Single Frequency ResponseSimplest Analyses – Single Frequency Response Basis in analytical chemistry Beer’s law response if isolated Protein treated as a solution  % helix, etc. is the unknown Standard in IR and Raman, Method: deconvolve to get components Problem – must assign component transitions, overlap -secondary structure components disperse freq. Alternate: uv CD - helix correlate to negative intensity at 222 nm, CD spectra in far-UV dominated by helical contribution Problem - limited to one factor, -interference by chromophores]

21 Single frequency correlation of De with FC helix

22 Problem of secondary structure definition No pure states for calibration purposes? ? ? helix sheet ? Need definition: Where do segments begin and end?

23 Next step - project onto model spectra–Band shape analysis Peptides as models - fine for a-helix, -problematic for b-sheet or turns - solubility and stability -old method:Greenfield - Fasman --poly-L-lysine, vary pH qi = aifa +bifb + cifc --Modelled on multivariate analyses Proteins as models - need to decompose spectra - structures reflect environment of protein - spectra reflect proteins used as models Basis set (protein spectra) size and form - major issue

24 Electronic CD spectra consistent with predicted helix contentElectronic CD for helix to coil change in a peptide Electronic CD spectra consistent with predicted helix content Note helical bands, coil has residual at 222 nm, growth of 200 nm band Loss of order becomes a question -- ECD long range sensitivity cannot determine remaining local order High temp “coil” Low temp helix 190 210 230 9

25 Ribonuclease A combined uv-CD and FTIR studyTyr92 Ribonuclease A combined uv-CD and FTIR study Tyr115 Tyr97 Tyr73 H1 H2 H3 Tyr76 Tyr25 124 amino acid residues, 1 domain, MW= 13.7 KDa 3 a-helices 6 b-strands in an AP b-sheet 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans) Ø Ø 6 b b sheet , 2 )

26 RibonucleaseA FTIR—amide I Near –uv CD Far-uv CD Loss of b-sheetLoss of tertiary structure Far-uv CD Loss of a-helix Spectral Change Temperature 10-70oC Stelea, et al. Prot. Sci. 2001

27 Ribonuclease A FTIR (a,b) Near-uv CD (tertiary) Far-uv CD (a-helix)PC/FA loadings Temp. variation FTIR (a,b) Near-uv CD (tertiary) Far-uv CD (a-helix) Temperature Stelea, et al. Prot. Sci. 2001 Pre-transition - far-uv CD and FTIR, not near-uv

28 Changing protein conformational order by organic solventTFE and MeOH often used to induce helix formation --sometimes thought to mimic membrane --reported that the consequent unfolding can lead to aggregation and fibril formation in selected cases Examples presented show solvent perturbation of dominantly b-sheet proteins TFE and MeOH behave differently thermal stability key to differentiating states indicates residual partial order

29 3D Structure of Concanavalin ADimer (acidic, pH<6) Tetramer (pH=6-7) Trp40 Trp109 Trp182 Trp88 High b-sheet structure, flat back extended, curved front Monomer only at very low pH, 4 Trp give fluorescence

30 Effect of TFE (50%) on Con A in Far and Near UV- CDFar UV-CD Near UV-CD Tertiary change with TFE - loosen Helix induced with TFE addition Xu&Keiderling, Biochemistry 2005

31 Dynamics--Scheme of Stopped-flow System- add dynamics to experiment Denatured protein solution Refolding buffer solution

32 Far UV (222 nm); [Con]f=0.2mg/ml Near UV (290 nm); [Con]f=2mg/mlStopped-Flow CD for Con A Unfolding with TFE (1:1) at Different pH Conditions Far UV (222 nm); [Con]f=0.2mg/ml Near UV (290 nm); [Con]f=2mg/ml pH=2.0 Xu&Keiderling, Biochemistry 2005

33 Zhang & Keiderling, Biochemistry 2006-lactoglobulin: a protein that goes both ways! Native state: -sheet dominant, but high helical propensity. Model: intramolecular  transition pathway as opposed to folding pathways from a denatured state. Zhang & Keiderling, Biochemistry 2006

34 Zhang & Keiderling, Biochemistry 2006          Lipid-induced Conformational Transition -Lactoglobulin 1. DMPG-dependent  transition at pH 6.8 Zhang & Keiderling, Biochemistry 2006

35 Charge-induced Lipid -- -Lactoglobulin Interaction          Charge-induced Lipid -- -Lactoglobulin Interaction Zhang & Keiderling, Biochemistry 2006 Increase DMPG, increases helix at expense of sheet

36 Stopped Flow Experiments : (pH 4.60)Vesicles (SUV) (DOPG, DMPG, DSPG) Vesicles (SUV) + BLG (0.2mg/ml) 5 Volume 1 Volume BLG (1.2mg/ml) CD: 222nm to monitor alpha-helix Fluorescence: filter with a 320nm cutoff ( Trp Tertiary Structure) 10-15 kinetic traces are collected and averaged Analysis：Multi-exponential function using Simplex Method: S(t)=a*t+b+∑i(ci Exp(-ki*t)) Ge, Keiderling, to be submitted

37 Stopped-Flow CD kinetic tracesDMPG Record at 222nm; N: trace without lipid vesicles; Traces are fitted to single-exponential function

38 Stopped-Flow fluorescence kineticsDMPG Total fluorescence >320nm; Each trace has been divided by kinetic trace without lipid vesicles; Traces are fitted to two-exponential function

39 Lipid bilayer insertion of -Lactoglobulin          Lipid bilayer insertion of -Lactoglobulin Fluorescence quenching ATR-FTIR orientation At pH 6.8 & 4.6, 4 & 6 nm blue shift in max. -helix Membrane surface Zhang & Keiderling, Biochemistry 2006

40 Summary: Lipid - b-Lactoglobulin InteractionNw Ns Unfolding Us Insertion Um Binding Zhang & Keiderling, Biochemistry 2006

41 Continued in Part b