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 now available that can separate subcomponents that overlap in the spectra of proteins. These facts have made practical biological systems amenable to studies 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.