Protein IR spectroscopy

3. Introduction
 During the last years the use of Fourier Transform Infrared spectroscopy (FTIR) to
determine the structure of biological macromolecules has dramatically expanded.
The complete three-dimensional structure of a protein at high resolution can be
determined by X-ray crystallography.
 The new technique of FTIR spectroscopy requires only small amounts of
proteins (1mM) in a variety of environments. Therefore, high quality spectra
can be obtained relatively easy without problems of background fluorescence,
light scattering and problems related to the size of the proteins. The
omnipresent water absorption can be subtracted by mathematical approaches.
Methods are nowavailable that can separate subcomponents that overlap in the
spectra of proteins. These facts have made practical biological systems
amenable tostudies by FTIR spectroscopy.

4. IR Spectroscopy –Active and In active
 A polar bond is usually IR-active .
 A non-polar bond in a symmetrical molecules will
absorb weakly or not at all.

5. IR spectroscopy is one of the most well established techniques for the analysis of protein
structure. In protein amino acids are covalently linked via amide(peptide) bonds, if absorb
in multiple regions of the mid-IR spectrum. By measuring amide bonds in protein chains
we can accurately quantifies an intrinsic components of every protein.
The N-H stretching region
N-H ……> 3300-3500cm-1
Primary amines gives two peaks
Secondary amines gives one peaks.
Tertiary amines gives no peaks.

6. Band Assignment
Amide Vibrations
The peptide group ,the structural repeat unit of proteins gives up to a characteristic
bands named amide A,B,I,II,….VII. The amide A band (about 3500 cm-1) and amide B
(about 3100 cm-1) originate from a Fermi resonance between the first overtone of
amide II and and the N-H stretching vibration. Amide I and amide II bands are two
major bands of the protein infrared spectrum. The amide I band (between 1600 and
1700 cm-1) is mainly associated with the C=O stretching vibration (70-85%) and is
directly related to the backbone conformation. Amide II results from the N-H
bending vibration (40-60%) and from the C-N stretching vibration (18-40%). This
band is conformationally sensitive. Amide III and IV are very complex bands
resulting from a mixture of several coordinate displacements. The out-of-plane
motions are found in amide V, VI andVIII.

7. The absorption of the protein backbone
Normal modes of the amide group
N-methylacetamide (NMA) is the smallest molecules that contains a trans peptide group. It has
therefore become the starting point for a normal mode analysis of polypeptide backbone vibrations.
If the CH3 group are regarded as point masses, the number of atoms of NMA is 6 and thus there are
12 normal modes.
IR Absorption spectroscopy
The energy of IR radiation corresponds to the small energy difference between vibrational and
rotational states in molecules. The IR region of the electromagnetic spectrum encompasses the
wavelength range from 0.78 to 1000µm.In IR spectroscopy the frequency of a band is usually
expressed in terms of wave numbers in units of cm-1.
IR spectral Region
1.Near IR
2.Middle IR
3.Far IR

8. Theory of Molecular Vibrations
Two kinds of fundamental vibrations
1.Stretching
2.Bending
Symmetric Stretching
Asymmetric Stretching
Scissoring
Wagging
Rocking
Twisting

9. Amino Acids
All amino acids exists as Zwitter ions at the Isoelecteric points due to the internal neutralization of the
acidic and the basic group in the molecules.
NH2CH2COOH…………› +NH3CH2COO-
Due to the insolubility of amino acids in common solvents,Infra-red spectrum of amino acids are
usually taken in the solid state. Amino acids in the form of zwitter ions do not show N-H stretching at
3200cm-1 but show broad band between 3130cm-1 and 3030cm-1 assigned to asymmetric stretching of
NH+3 group. Absorption depends on the structure of the amino acids. Hydrochloride of amino acids
containing NH3 group absorb between the 3145cm-1 and 3050cm-1.In zwitter ion two types of
vibrational modes of carboxylic ions.
The range of infra red radiations is 4000cm-1 to 667cm-1
Hooks law help to calculate approximately the value of stretching vibrational frequency of a bond.
The finger print region is below 1500cm-1

11. Amide A is with more than 95% due to the the N-H stretching vibration. This mode of vibration does not
depend on the backbone conformation but is very sensitive to the strength of a hydrogen bond. It has wave
numbers between 3225 and 3280 cm-1 for hydrogen bond lengths between 2.69 to 2.85 Å.
Amide I is the most intense absorption band in proteins. It is primilary governed by the stretching vibrations
of the C=O (70-85%) and C-N groups (10-20%). Its frequency is found in the range between 1600 and 1700
cm-1. The exact band position is determined by the backbone conformation and the hydrogen bonding pattern.
Amide II is found in the 1510 and 1580 cm-1 region and it is more complex than amide I. Amide II derives
mainly from in-plane N-H bending (40-60% of the potential energy). The rest of the potential energy arises
from the C-N (18-40%) and the C-C (about 10%) stretching vibrations.
Amide III, V are very complex bands dependent on the details of the force field, the nature of side chains and
hydrogen bonding. Therefore these bands are only of limited use for the extraction of structural information.

18. Summary
1. If the conventional FTIR spectroscopy can monitor protein
secondary structure features on the nanosecond to second
time scale.
2. The IR spectra of polypeptides and protein exhibit several
relatively strong absorption bands associated with the
CONH (amide) group, which change little in frequency and
intensity from one molecules to another.
3. Of all the amide modes of the peptide group the single most
widely used one in studies of protein secondary structure is
the amide I mode (1600-1700cm-1).
4. The type of secondary structure of polypeptide chains such
as alpha-helix,Beta-sheet,beta-turn and random coil.