1. Lecture 3: Absorption - UV/Visible/IR and CD Burning things to a crisp (as in the Bunsen Spectroscope) can tell us about the atoms contained in a molecule… But this doesn’t tell us anything about how the molecule is put together Fortunately, certain molecules absorb light in a characteristic way. Over the years, this has helped us identify and quantify biological molecules.

2. Absorption: Physical Basis Absorption occurs when the energy contained in a photon is absorbed by an electron resulting in a transition to an excited state http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm Since photon and electron energy levels are quantized, we can only get specific allowed transitions ~ 115 nm ~ 200 – 400 nm ~ 150-250 nm ~ 400 - 700 nm E=h (h = 6.626*10 -34 J  s)

3. Absorption: Lineshape  h ** So, our absorption spectrum should probably look like this: But they don’t…

4. Absorption: Lineshape This is because molecules are always rotating and vibrating. Each rotational or vibrational state slightly changes the energy of the transition. Distrubtion of these states is…a random walk. So the lineshape of our absorption spectra is… normally distributed

5. Absorption: Lineshape Absorption is an additive property, that is, the spectrum is the sum of the Gaussians associated with each transition This can make looking at complex solutions quite hard, especially if we are trying to say something about intensity

6. Absorption: Intensity The absorption efficiency of an analyte is affected by: The nature of the analyte The number of available microstates The solvent (sortof) The absorption efficiency of an analyte generally not affected by: Other (low conc.) solutes Concentration This makes absorption spectroscopy one of the few bioanalytical methods where the signal intensity is directly proportional to the concentration Temperature (within reason)

7. Absorption: The Beer-Lambert Law The Beer-Lambert law sortof has the wrong name… Pierre Bouguer (1698-1758) Johan Lambert (1728-1777) Astronomer: Light is diminished as it passes through the atmosphere. Mathematician, first to prove that  is irrational. No absorption coefficient. August Beer (1825-1863): Added absorption co- efficient and related to conc. in solution. Extinction coefficient Concentration Path length

8. Beer-Lambert Law Derivation (sortof) The Beer-Lambert law is a result of the fact that a photon has a constant probability of being absorbed as it travels through the sample. x y Absorption Total cross section of particles concentration Cross section of beam Populations with constant probabilities of ‘death’ decline exponentially

9. UV/Visible Spectroscopy: Instrumentation In absorption spectroscopy, we measure  as a function of wavelength The instrument we use to do this is called a UV/visible Spectrophotometer The Major Components Are: A light source A monochromator A detector A Sample Compartment

10. UV/Visible Spectroscopy: Light Sources Xenon, Mercury/Xenon Flash Arc-Lamps – light generated from Xe plasma Pure Xenon has very wide emission spectrum ~200 – 1200 nm Xenon/Mercury is blue shifted for more power in the UV region, used more often for sterilization

11. UV/Visible Spectroscopy: Light Sources Deuterium D 2 gas is discharged by contact with a high voltage tungsten cathode Continuous spectrum from ~150 nm - ~370 nm Usually used in conjunction with a tungsten/halogen source, which handles the visible spectrum

12. Monochromators The light sources we use produce continuous emission spectra. But we need single wavelengths, so… This is called the Czerny- Turner setup D is either a prism or a diffraction grating (almost always the latter these days B and C set up a beam that is at infinite focus E refocuses one wavelength on slit F Different wavelengths can be focused on F by rotating D or E (it’s almost always D).

13. Monochromators: Defraction Gratings Diffraction gratings are a surface with closely spaced parallel lines These can be semi-transparent gratings or ridged mirrors After passing though a slit (or bouncing off a ridge) the angle at which the light leaves is given by wavelength! distance between slits diffraction order

14. Sample Compartments/Holders These days, sample compartments are designed to accept accessories… The sample itself is held in a cuvette, usually plastic or quartz:

15. The detector Silicon diode: Basically a solar cell – light ionizes n-doped (phosphate) silicon, placing the electrons in the conduction band (i.e. having a voltage). Wide wavelength range, less sensitivity

16. Photomultiplier In photomultipliers, light hits a photocathode, releasing a small number of electrons, which are then made to collide with a series of dynodes, each more positive than the last Each collision produces more and more activated electrons Sensitive, but noisy. Pretty much needed for low energy (IR) photons

17. The Whole Instrument Light Source Czerny-Turner Sample Detector

18. UV/Visible - Applications UV/visible absorption spectroscopy may not be a ‘new and sexy’ method, but it has one advantage: The signal is proportional to the concentration. Consequently, it is most commonly used for concentration measurements or validation: Protein concentration with dyes (Bradford) and without (A 280 - Tryptophan) Purity of protein or nucleic acid preps (A 260 /A 280 )  = 5,579 M -1 cm -1 at 278 nm

19. UV/visible: Applications UV/visible is still used in current research, especially for heme-containing proteins, which have absorbance in the Soret region that is sensitive to the state of the protein 3d shell of Fe 2+ has 6 electrons High SpinLow Spin

20. UV/Visible Applications This paper looks at iron-sulfur clusters in a native and a mutant protein C196S Mutant lacks broad absorption band between 400- 600 nm which is diagnostic of an 2Fe-2S cluster JBC (1998)Vol. 273, No. 35;28 pp. 22311–22316

21. Time-resolved UV/visible The main protein signal (at 280 nm) doesn’t change much with protein folding/activity But Soret region absorption does (cytochrome P450 cam )… Spolitak T, Dawson JH, Ballou DP (2005) J. Biol. Chem. 280 (21): 20300-20309 2005

22. Circular Dichroism (CD) So far, we’ve gotten our light down to a single wavelength, but it’s not polarized Plane polarized (i.e. a laser) Circularly polarized

23. CD and Chirality Chiral molecules absorb left and right circularly polarized light differently L RL R L R L L RL R L R L This difference, which can be expressed as which is the circular dichroism Usually expressed as molar elipticity:

24. CD and Proteins In proteins there are a number of circularly dichroistic electronic transitions that are of use: n  * (220 nm)  * (190 nm) dipole orientation of F, Y, C and W (250-300 nm) But these are all weakly dichroistic. In proteins  ~.0001

25. CD Spectra of Proteins In CD Spectra, we measure  as a function of wavelength: -ve Peaks at 208 and -222 nm =  -helix -ve, broad Peak at -218 nm =  -sheet +ve at 212, -ve at 190 nm =  -turn Problem: Overlap!!!

26. CD Data Analysis: CD data can provide very specific information about secondary structure. We should be able to convert the intensity of the peak at 222nm, for example, into ‘% alpha helix’. But what if there’s overlap with the  sheet peak? The most common solution is to use a ‘basis set’ of archetypal spectra, adding them together in a way that most closely matches the observed spectrum. Helix 35% Sheet 22% Turn 10%

27. CD is Noisy! Not only is CD a very small signal, but in the far UV region, there are lots of sources of noise One of these is the production ozone (O 3 ) in the sample compartment. This is why most CD instruments are flushed with N 2 HTC Noise/Signal (effectively)

28. CD Instrumentation So we need most of the same things as for a UV/visible: Lamp (Xe or Xe/Hg) Monochromator (Czerny- Turner) Detector (photomultiplier) But we also need: Polarizer Electro-optic modulator Sample compartment

29. Applications CD compliments other more general methods of monitoring (un)folding. Babu KR, Douglas DJ, (2000) Biochemistry 39 (47): 14702-14710 Tertiary structure Secondary Structure

30. Time-resolved CD CD compliments other more general methods of monitoring (un)folding. Dartigalongue T, Hache F, Chirality 18 (4): 273-278 MAY 5 2006 Kaushik JK, Ogasahara K, Yutani K, J. Mol. Biol., 316 (4): 991-1003 MAR 1 2002

31. Time-resolved CD CD compliments other more general methods of monitoring (un)folding. J. Mol. Biol., 372 (1): 236-253 2007