spectroscopic methods for structural analysis of biological macromolecules d. krilov 20.10. 2008
TRANSCRIPT
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