To be covered (and why) Spectroscopy of Proteins. UV-Vis Absorption. UV-Vis Absorption. Spectra

Transcription

1 To be covered (and why) Spectroscopy of Proteins General considerations UV-Vis Absorption quantitation Fluorescence hydrophobicity Foldedness FT-Infrared Foldedness ircular Dichroism Foldedness NMR (a little) UV-Vis Absorption UV-Vis Absorption Electronic transitions Proteins Vis Metal ions (d-orbitals) ofactors (conjugated double bonds) UV Aromatics Peptide bond excited 2.. excited 1 hν ground (and ν = c/λ) Vibrational levels Electronc levels When hν = ΔE a transition can occur Absorbance is described by Beer s Law: A λ = ε λ lc (extinction, pathlength & concentration) Vibrational transitions are in the IR or microwave region Electronic transitions are in the UV or Vis but involve different vibrational levels Spectra A spectrum is basically a plot of A or ε versus λ or ν or energy. A peak occurs where A λ or ε λ is large For proteins, peaks occur at 280nm, ε 280 ~ 1/mg ml - Trp & Tyr nm, ε= large - n-π* transitions involving the peptide bond Mostly useful for quantitation Absorbance Trp Phe Wavelength (nm) Tyr Where does the energy go? The energy is dissipated as heat or re-emitted - Fluorescence Intrinsic fluorescence Trp Bound cofactors Noncovalent Probes ANS cis-parinaric acid NH SO 3-1-Anilino-8-naphthalene sulfonate (ANS)

2 Other Sources of Fluorescence ovalently-attached probes Usually to ys-sh, R-NH 2 See Molecular Probes catalog (thiol-reactive probes, amine-reactive probes) Fuse a gene for Green Fluorescent Protein (GFP) to your gene Several fluorophores (colors) are available. Excitation and Emission Spectra ould set detector as emission λ max and scan excitation λ (measure emission) ould set excitation at ε max and scan emission λ Maxima are different because of loss of heat (vibrational levels) Absorption depends on ε, ext. coeff.; emission depends on ϕ, quantum yield λ max - λ max = Stokes Shift Fluorescence is altered by environment Quenching Mechanisms S 2 S 1 S 0 ground state solvation A B S 1 excited state solvation S 0 Processes time scale A thermalization ~10 12 s -1 B solvent relaxation <10 11 s -1 emission ~10 8 s Polar solvents can reorganize to stabilize excited state, which destabilizes ground state red shift of fluorescence 2. Polar solvents can quench Absorb excitation radiation (apparent quenching) Absorb emitted radiation by another cmpnd Abs spectrum overlaps with emission spectrum an be used for FRET to get distances ontact quenching Pass the energy to attached molecule Dissipates it as heat ollision quenching Excited fluor collides with a molecule auses relaxation of the fluor without emission Illustration Molecular Vibrations Solvents Octanol Butanol Propanol Ethanol Methanol Ethylene glycol Relative Intensity Emission wavelength (nm) Note quenching & shift Fluorescence ns 25 ns Wavelength (nm) Time dependence of shift So, fluorescence wavelength & intensity (quantum yield) report on the environment of the fluorophore m = µ = m 1 m 2 m 1 + m 2 ν = 1 k 2π µ x = asin k µ E = hν = h k 2π µ F = ma F = kx = m d 2 x dt 2 x = asin(2πνt)

3 Quantum mechanically: H = E More than 2 atoms? Potential E U x = kx U = 1 2 kx 2 h 2 d 2 ψ 8π 2 m dx + 1 kx 2 ψ = Eψ 2 2 h E n = ( n + 1 2) k 2π µ Kinetic E = h( n + 1 2)ν 0 p = mv T = 1 2 mv 2 T = p2 2m but : p = i h d 2π dx Normal Modes atoms move in straight lines atoms move in phase the center of mass does not move Molecule with N atoms has 3N - 6 normal modes 3N - 5, for linear molecules Still too complex Let s focus on characteristic vibrations R O NHR' R H O NHR' R" N R'" FTIR Interpretation Amide I & II regions H-bonding & torsion angles in α-helix and β-sheet perturb the vibrational frequency of the amide =0 differently. The IR spectrum of the Amide regions should be a linear combination of the spectra of helix and sheet Beware In soluble proteins, Amide I occurs at lower wavenumbers, approximately 1650 to 1655 cm -1. Helical proteins associated with membranes, from 1656 to 1658 cm -1 Highly solvent exposed helices in D2O, as low as 1644 cm -1. Beware! Protein IR Spectroscopy omplex - people are trying to assign many vibrations - difficult. Use in combination wit other tools. The trick is to get a spectrum Use D 2 O to avoid water vibrations Use FT IR to average many scans and improve signal:noise

4 FTIR Interferometer Measure frequencies by mutual interference Highfreq waves go in & out of phase at high freq Each beam of light is compared with itself It interferes with itself at a characteristic frequency Interferogram Interferogram Fourier Transform See NMR Tutorial pure sine wave pure sine wave Exponentially Decaying intensity Exponentially Decaying intensity Two Two time frequency Refractive Index Speed of light in a vacuum is c = 2.99x cm/s (Not just a good idea, it s the LAW) Elsewhere, v = c/n, where n = index of refraction auses misbehavior A thought experiment Huygens & Snell s Law Medium has n = n i Transparent block with n = n r > n i How long does it take light to go B->D, compared to A->, considering it travels slower along A? X i normal A r v = c/n r B v = c/n i Same time; it cheats and takes a shortcut: r < i so that A < BD D

5 Snell s Law: sini v i n r = = sin r v r n i Light travels A at vi = c/ni in the same time that it travels BD at vr = c/nr Each must take time = distance/v A/(c/ni) = BD/(c/nr) The two triangles share the hypotenuse AD BD = ADsini; A = ADsinr light bends Plane Polarized Light an be decomposed into the sum of two oppositely-polarized circularly polarized beams Plane Polarized light Wave has two components Electric vector Magnetic vector Treat like a complex plane and ignore the imaginary one Filter out waves with vectors in transverse directions, or Have a polarized source Electric vectors are contained in a plane Plane Polarized Light What happens of a substance has a different refractive index for right- and left-circularly polarized light? A right-handed circularly-polarized beam The plane rotates optical rotation Stereo pair of two circularly-polarized beams adding to a plane Rotation of the Plane The components no longer meet at the top and bottom of their cycles They still meet, and cancel each other out except in one plane, but a different plane a polarization of source Plane Polarized Light? What happens of a substance has a different extinction coefficient for right- and leftcircularly polarized light? The two vectors are of different magnitudes they never cancel rotation of plane of polarization by retarding the leftturning component. The light becomes elliptically polarized 5

6 ircular Dichroism. Need: an Optical Transition + an Asymmetric Structure The principle is that ε L and ε R are different, and ellipticity is related to (ε L - ε R ) λ. oncentration and pathlength are important, so (A L - A R ) λ : may be used: ψ λ = (A L - A R ) λ in degrees. an be decomposed into specific ellipticity: [ψ] λ = ψ λ /l c Or, for polymers, mean ellipticity [] λ, meaning the average over the number of monomers: [θ] λ = M o [ψ] λ /100 which could also be written [θ] = 100[ψ] λ /l c, in which the primes signify that pathlength is in cm and concentration is in moles of monomer per liter. With a little bit of fiddling, you can show that [θ] λ = 3300(ε L - ε R ) λ. aromatic amino acids (260nm-280nm) dominated by tryptophan, with a contribution from tyrosine only if tightly packed (planar molecules are symmetrical) the peptide bond (180nm-~235nm) Each 2 Structure has different D spectrum λ = F i X iλ Secondary Structure NMR Spectroscopy Elipticity [T] ( cm 2 /0.1 mole x 10 3 ) α helix β random coil Ellipticity at any λ is a weighted aveg of helix, beta, turn & coil: X λ = f H X λh + f β X λβ + f t X λt + f R X λr For folding kinetics, watch ellipticity at 222nm Online tutorial at ~garysmith/nmr.dir/nmr1.htm Wavelength (nm)