SPECTROSCOPIC METHODS FOR STRUCTURAL ANALYSIS OF BIOLOGICAL MACROMOLECULES

D. Krilov

20.10. 2008.

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

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)

6r

BrU

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

repulsive interactions: at very short distances the repulsive forces predominate - forces between atomic nuclei and between electronic clouds:

for numerical calculations the potential is:

0rreArU

12r

ArU

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

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

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

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

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

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 (+)

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)

Electromagnetic spectrum

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=h0

energy is transferred to matter in quanta

The concept of wavepacket (quantum of energy)

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

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

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

2

2maxvm

h A. Einstein 1905.

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

22 cmh e

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 ...

vn EEh

molecule will emit photon if it is inexcited state, i.e. with excess ofenergy

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.

Attenuation of electromagnetic radiation in matter

Due to interaction of photons with molecules the intensity of the beamis decreasing along its path throughthe sample

- I = I2-I1= k I1 x - dI = k I dx

I1 I2

I0I

x

I

I

x

dxkI

dIdxk

I

dI

0 0

xkeIxI 0

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 / I0

absorbance A = - log I = log I0 / I

molar absorptioncoefficient

concentration

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.

Spectroscopic techniques

Absorption Emission

I0 It

partialabsorption

transmission

excitation

emission

I0

Ie

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)

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