1. SPECTROSCOPIC METHODS FOR STRUCTURAL ANALYSIS OF BIOLOGICAL MACROMOLECULES D. Krilov 20.10. 2008.

2. Interactions in biological macromolecules  Van der Waals's forces; hydrogen bond; hydrophobic interactions; ionic bonds  interactions between atomic groups in macromolecule, between macromolecule and smaller molecules or macromolecule and water  these interactions are of electrostatic nature  they are about 20 times weaker than covalent bond  they determine the secondary and tertiary structure of macromolecules

3. Van der Waals's forces  attractive interactions between molecules with closed shells (even number of electrons in outer shell):  a) nonpolar molecules - dispersion interactions between transient dipoles induced by fluctuation of electrons  b) polar molecules - interactions dipole-charge, dipole- dipole, induced dipole-dipole, induced dipole-induced dipole  potential energy  the bond is multi directional and unsaturated  (one molecule can form several such bonds with surrounding molecules)

4. Energies of attractive interactions  at 25°C the average energy of interaction is - 0,07 kJ mol -1 (kinetic energy is 3,7 kJ mol -1 )  energy of interaction is - 0,8 kJ mol -1  energy of interaction is about - 5 kJ mol -1 ; it depends on molecule polarizability

5.  repulsive interactions: at very short distances the repulsive forces predominate - forces between atomic nuclei and between electronic clouds:  for numerical calculations the potential is:

6. Hydrogen bonds  attractive interactions between molecules with closed shells, of specific structure: A - H ··· B  A and B are strongly electronegative elements (usually N, O, F)  B must have a free electron pair  due to electronegativity of A atom, the hydrogen atom tends to localize between A and B; in that way H becomes partially positive and B partially negative  this bond is unidirectional and saturated  (one hydrogen atom can form only one hydrogen bond)  bond energy is about 20 kJ mol -1

7. Examples of hydrogen bond  a) between atoms in adjoining molecules  the bond is stronger when the atoms are aligned  hydrogen bond in biological molecules:  between two amino acids in polypeptide chain,  between pairs of bases in nucleic acids 0,2 nm

8.  b) in water  each molecule capable of creation the hydrogen bond with another molecule, creates such bond also with molecules of water  that is the reason why the hydrogen bond between two molecules becomes weaker when they are dissolved in water  among water molecules there is a network of hydrogen bonds which is responsible for the specific properties of water

9.  the hydrogen bonds exist between the surface of macromolecule and surrounding water molecules  the layer of partially immobilized molecules of water arround a macromolecule is called hydration shell elastine

10. Hydrophobic interactions  the hydrophobic groups are forced by water to stick together in order to minimize their influence on hydrogen bonding network  this assembling is described as hydrophobic bond, but actually these are the repulsive interactions between molecules of water and hydrophobic groups  such ordering diminishes the total energy and increases the entropy of the system

11. Ionic bonds  a) in macromolecules  ionic electrostatic interactions are present between charged groups; they are strong in the absence of water molecules  b) in aquaeous solutions  ionic interactions are less strong and ionic bonds are weak, especially when there are dissolved salts in water  enzyme (-) is bound to the substrate (+)

12. ELECTROMAGNETIC RADIATION  electromagnetic waves – communication with the outer world: sight, the sense of heat, communication facilities (radio, TV, cell phones …)  interaction with matter: information about structure and dynamics of molecules; conformations of macromolecules and their interaction with environment  the sources: natural (atoms, molecules, cosmic rays, stars); artificial (aerials, lamps, X-ray tube, cobalt bomb)

13. Electromagnetic spectrum

14. Interaction of electromagnetic field with matter  it is explained by particle nature of radiation: wavepacket – photon (Einstein 1905.)  natural and artificial sources of radiation are not simple harmonical oscillators – the emitted waves are in the narrow range of frequencies arround  0 :   =  0  ,  <<  0  the interference of the waves of close frequencies results in energy localization in the form of the wave packet; its energy is E=h 0  energy is transferred to matter in quanta

15. The concept of wavepacket (quantum of energy)

16. Nonionizing interactions  after absorption of incident photon, atom or molecule is raised to higher energy state or there is an increase in overall translational motion - heating of the matter  elastic scattering of incident photon at atom

17. Elastic incoherent scattering of photon – Compton's effect  collision of photon with atom results in ejection of electron from outer shell; the scattered photon has lower energy and different direction  the recoil electron can induce further ionizations  the remaining cation is relaxed by emission of secondary photon  the interaction is more probable for photons with energy much higher from the ionization energy of electron in atom

18. Absorption of photon – photoelectric effect  the incident photon which collides with atom is completely absorbed and electron is ejected from an inner shell  the recoil electron can induce further ionizations  the remaining cation is relaxed by emission of secondary photon the probability of interaction is higher for the photons with lower energy A. Einstein 1905.

19. Pair production  in the vicinity of heavy nucleus photon with energy higher than 1 MeV can be transformed into the pair of particles: electron - positron  the heavy nucleus takes over the part of photon 's momentum

20. Spectroscopy  the methods are based on interaction of electromagnetic radiation with matter  the molecule will absorb photon if its energy is equal to energy difference of two energy states in molecule:  the properties of molecule are changed: electrons distribution, electric dipole momentum, magnetic momentum of nucleus or electron... molecule will emit photon if it is in excited state, i.e. with excess of energy In each spectroscopy method the photons will interact with matter if their energy corresponds to the energy differences determined by the structure and properties of molecular pattern of the sample.

21. Attenuation of electromagnetic radiation in matter Due to interaction of photons with molecules the intensity of the beam is decreasing along its path through the sample  -  I = I 2 -I 1 = k I 1  x  - dI = k I dx I1I1 I2I2 I0I0 I xx

22.  k( ) is attenuation coefficient which depends on the medium and wavelength of radiation  when the radiation is passing through the solution:  k (,c) =  ( ) c  transmittance T = I / I 0  absorbance A = - log I = log I 0 / I molar absorption coefficient concentration

23. Characteristical spectral parameters  Spectrum is the distribution of spectral radiancy ( I ) (or absorbance, or molar absorpton coefficient…) over energy (or wavelength, or frequency, or wave number)  The line position reflects the transition energy between two states  The line intensity is the measure of the number of equal transitions  The line width depends on dynamics of the environment of investigated molecule; the higher is the number of collisions with other molecules, the shorter is the lifetime of excited state; the spectral line is broadened  The ground state of molecule is the state with minimal energy; in all spectroscopy ranges it is predominantly populated. That means that the process of absorption of photon is always possible.

24. Spectroscopic techniques  Absorption  Emission I0I0 ItIt partial absorption transmission excitation emission I0I0 IeIe

25. Basic spectroscopic methods in biology and medicine  Absorption spectroscopies:  1. Optical or electron spectroscopy – electron transitions between molecular orbitals; the change in electron distribution; spectra in visible and ultraviolet range (100-700 nm)  2. Infrared spectroscopy – transitions between vibrational states; change in the value of electric dipole momentum; spectra in infrared range (800-10000 nm)  3. Electron spin resonance– transitions between electron spin states in external magnetic field; the change of magnetic spin momentum of electron; spectra in microwave range (1-10 cm)  4. Nuclear magnetic resonance - transitions between nuclear spin states in external magnetic field; the change of magnetic spin momentum of nucleus; spectra in radiowave range (1 – 10 m)

26.  Emission spectroscopies:  Fluorescence – molecules are excited to higher energy state by ultraviolet or laser radiation; in the process of relaxation to the ground state they emit the radiation in visible range; molecules or supramolecular structures which don't possess intrinsical fluorophores are labeled by covalently bonded fluorescence probes