Electronic Spectroscopy Chem 344 final lecture topics Time out - PowerPoint PPT Presentation

Electronic Spectroscopy Chem 344 final lecture topics

Time out — states and transitions Spectroscopy — transitions between energy states of a molecule excited by absorption or emission of a photon h n = D E = E i - E f 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

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 z B | E E || z } Polarization B || x y k || y x n = c/ l l Frequency Wavelength

Properties of light – probes of structure • Frequency matches change in energy, type of motion  E = h n , where n = c/ l (in sec -1 ) • Intensity increases the transition probability —  I ~ e 2 – where e is the radiation Electric Field strength Linear Polarization (absorption) aligns with direction of dipole change — (scattering to the polarizability)  I ~ [ dm / d Q] 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 IR of 1.2 (Absorbance) vegetable Absorbance .8 oil .4 n l 0 4000 3000 2000 1000 -1 Frequency (cm )

Optical Spectroscopy - Processes Monitored UV/ Fluorescence/ IR/ Raman/ Circular Dichroism Analytical Methods Diatomic Model Excited Absorption UV-vis absorp. State h n = E grd - E ex (distorted & Fluorescence . geometry) move e - (change electronic state) high freq., intense Ground CD – circ. polarized State (equil. n 0 n S Fluorescence geom.) absorption, UV or IR h n = E ex - E grd Raman – nuclei, Raman: D E = h n 0 -h n s inelastic scatter very low intensity = h n vib IR – move nuclei Infrared: D E = h n vib low freq. & inten. Q molec. coord. 0

Opt ptica ical l Spe pectrosc troscopy opy – Ele lectronic, tronic, Examp ample le Abs bsorpti orption on an and d Flu luor ores escen cence ce Essentially a probe technique sensing changes in the local environment of fluorophores What do you see? (typical protein) Intrinsic fluorophores e (M -1 cm -1 ) eg. Trp, Tyr Change with tertiary structure, compactness Amide absorption broad, Intense, featureless, far UV ~200 nm and below

Circular Dichroism • Most protein secondary structure studies use CD • Method 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

Circular Dichroism CD is polarized differential absorption D A = A L - A R 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 , PO 2- , C=O in IR Coupled transitions between amides along chain lead to distinctive bandshapes

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

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

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

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

b -sheet cross-strand H-bonding

Anti-parallel b -sheet (extended strands)

Polypeptide Circular Dichroism ordered secondary structure types a -helix De b -sheet turn Brahms et al. PNAS, 1977 l poly-L-glu( a , ____ ), poly-L-(lys-leu)( b, - - - -), L-ala 2 -gly 2 (turn, . . . . . ) Critical issue in CD structure studies is SHAPE of the De pattern

Large electric dipole transitions can couple over longer ranges to sense extended conformation Simplest representation is coupled oscillator n    π    ) m a    m  m   T ab  R T ab a b   m b 2 c De  Dipole coupling results in a e L -e R l derivative shaped circular dichroism Real systems - more complex interactions - but pattern is often consistent

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

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

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

Simplest 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]

Single frequency correlation of De with FC helix  (222 nm) vs FC helix  (193 nm) vs FC helix De at 222nm/193 nm 10 0 0 20 40 60 80 FC helix [%]

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

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  i = a i f a +b i f b + c i f c -- 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

Electronic CD for helix to coil change in a peptide Electronic CD spectra consistent with predicted Note helical bands, coil has residual at 222 nm, growth of 200 nm band helix content 5 0 0 0 0 4 0 0 0 0 Loss of order becomes a question -- ECD long range sensitivity cannot 3 0 0 0 0 determine remaining local order 2 0 0 0 0 1 0 0 0 0 High temp “coil” 0 - 1 Low temp helix - 2 - 3 190 210 230 1 2 2 2 2 2 2 2 9 0 1 2 3 4 5 6 0 0 0 0 0 0 0 0 Ellipticity Wavelength (nm)

Tyr92 Ribonuclease A Tyr115 Tyr97 Tyr73 combined uv-CD H1 and FTIR study 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 b b  6 sheet • 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans) , 2 )

0.06 RibonucleaseA FTIR 0.05 0.04 Absorbance 0.03 0.02 FTIR — amide I 0.01 Loss of b -sheet 0.00 1720 1700 1680 1660 1640 1620 1600 Wavenumber (cm -1 ) 0 -2 Near – uv CD -4 Ellipticity (mdeg) -6 -8 Loss of tertiary -10 structure -12 -14 Near-UV CD -16 260 280 300 320 Wavelength (nm) Far-uv CD 5 Ellipticity (mdeg) Loss of a -helix 0 -5 Spectral Change -10 Far-UV CD Temperature 10-70 o C -15 190 200 210 220 230 240 250 Stelea, et al. Prot. Sci. 2001 Wavelength (nm)

Ribonuclease A -6.4 1.0 FTIR -6.8 0.5 PC/FA loadings 2 ) Temp. variation C i1 (x10 -7.2 0.0 -7.6 -0.5 FTIR ( a,b ) -8.0 -1.0 -5 10 -7 5 -9 Near-uv CD 0 C i2 C i1 Near-UV CD -11 -5 -13 (tertiary) -10 -15 -17 -15 -10 5 Far-uv CD 0 -11 -5 ( a -helix) Far-UV CD C i1 C i2 -10 -12 -15 -20 -13 -25 Temperature -30 0 20 40 60 80 100 Stelea, et al. Pre-transition - far-uv CD and FTIR, not near-uv Prot. Sci. 2001

Changing protein conformational order by organic solvent TFE 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

3D Structure of Concanavalin A Dimer (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

Effect of TFE (50%) on Con A in Far and Near UV- CD Far UV-CD Near UV-CD Tertiary change Helical Content Helix induced with pH=7 43% with TFE - loosen TFE addition pH=2 57% Xu&Keiderling, Biochemistry 2005

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