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  by Andreas Jabs (Andreas has left our group. So, direct enquiries to Juergen Suehnel).

Introduction
           Basic principles of infrared (IR) absorption
           Fourier Transform Infrared (FTIR) spectroscopy

Band assignments
         Amide vibrations
         Amino acid side chain vibrations

Secondary structure of peptide model compounds
         Beta sheet structures
         Helical structures
                     alpha helix
                     310-helix
         Turn structures

Secondary structure in proteins
         Deconvolution of the amide I band
         Second derivative spectra and curve fitting



Introduction

During the last years the use of Fourier Transform Infrared spectroscopy (FTIR) to determine the structure of biologicalmacromolecules has dramatically expanded.
The complete three-dimensional structure of a protein at high resolution can be determined by X-ray crystallography. This technique requires the molecule to form a well ordered crystal which is not possible for all proteins. An alternative to X-ray crystallography is multidimensional nuclear magnetic resonance (NMR) spectroscopy. Using NMR spectroscopy structures of the proteins can be determined in solution. The interpretation of the NMR spectra of large proteins is very complex, so its present application is limited to small proteins (~15-25 kDa). These limitations have led to the development of alternative methods that are not able to generate structures at atomic resolution but provide also structural information on proteins (especially on secondary structure). These methods include circular dichroism (CD) and vibrational (infrared and RAMAN) spectroscopy.
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.
 
 

Basic principles of infrared (IR) absorption

We will focus on very few aspects here, because many textbooks present excellent descriptions of the basis of IR spectroscopy (see for example Campbell & Dwek, in Biological Spectroscoy, Benjamin Cummings, Menlo Park, CA 1984 and Brey, Physical Chemistry and its Biological Applications, Academic Press, New York, 1984, p.133).
IR spectroscopy is the measurement of the wavelength and intensity of the absorption of infrared light by a sample. Infrared light is energetic enough to excite molecular vibrations to higher energy levels.


Electromagnetic spectrum

frequencyrange (Hz) wavelength range type of radiation type of transition
1020 - 1024 10-12 - 10-16 m gamma rays nuclear
1017 - 1020 1 nm - 1 pm x-rays inner electrons
1015 - 1017 400 - 1 nm ultraviolet light outer electrons
4.3x1014 - 7.5x1014 700 - 400 nm visible light outer electrons
1012-1014 2.5 um - 700 nm infrared light vibrations
108 - 1012 1 mm - 2.5 um microwaves rotations
100 - 108 108 - 1 m radio waves spin flips

The infrared spectra usually have sharp features that are characteristic of specific types of molecular vibrations, making the spectra useful for sample identification.

Table of characteristic IR bands

X-H vibrations bond wavenumbers (cm-1)
hydroxyl O-H 3610-3640 
amines N-H 3300-3500
aromatic rings C-H 3000-3100
alkenes C-H 3020-3080
alkanes C-H 2850-2960
triple bonds   2500-1900
double bonds   1900-1500
deformation/heavy atoms   1500-

For a molecule of N atoms, 3N-6 fundamental vibrations (or normal modes) exist (3N-5 if the molecule is linear). Therefore, for the linear CO2 molecule 4 normal modes have to be expected.

Normal modes for CO2

cm-1        IR  RAMAN
stretching (sym.)


->          <-
O==C==O

 
1340 - +
stretching (asym.)


->   <-  <-
O==C==O

 
2349 + -
deformation
/|            /|
O==C==O
\|
667 + -
deformation


+     -     +
O==C==O

 
667 + -


 

Fourier Transform Infrared (FTIR) spectroscopy

To use the Fourier Transform Infrared Spectroscopy, a continuum source of light (such as a Nernst Globar) is used to produce light over a broad range of infrared wavelengths. Light coming from this continuum source is split into two paths using a half-silvered mirror; this light is then reflected from two mirrors back onto the beamsplitter, where it is recombined. One of these mirrors is fixed, and the second is movable. If the distance from the beamsplitter to the fixed mirror is not exactly the same as the distance from the beamsplitter to the second mirror, then when the two beams are recombined, there will be a small difference in the phase of the light between these two paths. Because of the "superposition principle" constructive and destructive interference exist for different wavelengths depending of the relative distances of the two mirrors from the beamsplitter.
It can be shown that if the intensity of light is measured and plotted as a function of the position of the movable mirror, the resultant graph is the Fourier Transform of the intensity of light as a function of wavenumber . In FTIR spectroscopy , the light is directed onto the sample of interest, and the intensity is measured using an infrared detector. The intensity of light striking the detector is measured as a function of the mirror position, and this is then Fourier-transformed to produce a plot of intensity vs. wavenumber.
As radiation source a Michelson Interferometer is used (see the drawing below).  
 



It is necessary to increase the sensitivity somehow, because the absorption due to one monolayer of molecules typically results in a change in intensity of only about one part in 105. For semiconductors, one way of increasing the sensitivity is to use multiple internal reflection. In this technique, the edges of the sample are polished, and the light is sent in at an angle. The light bounces around inside the sample, making about 30-50 bounces. This increases the sensitivity by about a factor of 30-50, making it possible to measure the absorption of less than one monolayer of molecules on a surface.

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Band assignments

Amide vibrations

The peptide group, the structural repeat unit of proteins, gives up to 9 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.

click to enlarge


 

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 wavenumbers between 3225 and 3280 cm-1 for hydrogen bond lengths between 2.69 to 2.85 Å, (Krimm & Bandekar Adv Protein Chem 1986;38:181-364).
Amide I is the most intense absorption band in proteins. It is primilary goverend 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.

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Amino acid side chain vibrations

The presence of bands arising from amino acid side chains must be recognized before attempting to extract structural information from the shapes of amide I and amide II bands. The contribution of the side chain vibrations in the region between 1800 and 1400 cm-1 (amide I and amide II region) has been thoroughly investigated by Venyaminov & Kalnin 1990 (Biopolymers 1990;30(13-14):1243-57). Among the 20 proteinogenous amino acids only 9 (Asp, Asn, Glu, Gln, Lys, Arg, Tyr, Phe, His) show a significant absorbance in the region discussed above. The contribution of the different amino acid side chains were fitted by a sum of Gaussian and Lorentzian components.
 
 

    AS vibration   cm-1 A0
(l/mol/cm)
FWHH
(cm-1)
surface 
(x10-4 l/mol/cm)
Asp -COO st as pH>pK (~4.5) 1574 380 44 5.5
  -COOH st  pH<pK (~4.5) 1716 280 50 4.1
Glu -COO st as  pH>pK (~4.4) 1560 470 48 7.1
  -COOH st pH<pK (~4.4) 1712 220 56 3.6
Arg -CN3H5+ st as   1673 420 40 4.3
                  st s   1633 300 40 3.6
Lys -NH3+ bd as   1629 130 46 1.8
             bd s   1526 100 48 1.3
Asn -C=O st   1678 310 32 2.7
  -NH2 bd   1622 160 44 2.5
Gln -C=O st   1670 360 32 3.1
  -NH2 bd   1610 220 44 3.5
Tyr ring-OH pH<pK (~10) 1518 430 8 1.0
  ring-O pH>pK (~10) 1602 160 14 0.7
      1498 700 10 2.5
His ring   1596 70 14 0.3
Phe  ring   1494 80 6 0.2
 terminal             
  -COO st as   1598 240 47 3.5
  -COOH st    1740 170 50 2.1
  -NH3+ bd as   1631 210 54 3.8
  bd s   1515 200 60 4.3
  -NH2 bd   1560 450 46 7.5

frequency, absorbance at the maximum (Ao), full width at half height (FWHH), surface of Gaussian band
st=stretching vibration
bd=bending
s=symetrical
as=asymetrical

(according to Venyaminov & Kalnin, Biopolymers 1990;30(13-14):1243-57)
 

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Secondary structure of peptide model compounds

A large number of synthetic polypeptides has been used for the characterization of infrared spectra for proteins with a defined secondary structure content. For example, polylysine may adopt both beta-sheet or alpha-helical structures in dependence on temperature and pH of the solution. Experimental and theoretical work on a large number of synthetic polypeptides has provided insights into the variability of the frequencies for particular secondary structure conformations (Krimm & Bandekar, Adv Protein Chem 1986;38:181-364  )

Beta sheet structures
(beta strand)
The frequencies of the main absorption bands from synthetic polypeptides adopting an antiparallel chain structure have been compiled by Chirgadze & Nevskaya (Biopolymers 1976 Apr;15(4):637-48). From these data it this follows, that the amide I absorption is primarily determined by the backbone conformation and independent of the amino acid sequence, its hydrophilic or hydrophobic properties and charge. The average frequency of the main component is about 1629 cm-1 with a minimum of 1615 cm-1 and a maximum of 1637 cm-1. The average value for the second frequency is 1696 cm-1 (lowest value 1685 cm-1). The parallel beta  sheet structure that is not common in synthetic polypeptides leads to an amide I abosrption near 1640 cm-1
 

Helical structures

The alpha-helix: For alpha-helical structures the mean frequency was found to be 1652 cm-1 for the amide I and 1548 cm-1 for the amid II absorptions (Chirgadze & Nevskaya, Biopolymers 1976 Apr;15(4):637-48). The half width of the alpha-helix band depends on the stability of the helix. For the most stable helices, the half-width of about 15 cm-1 corresponds to a helix-coil transition free energy of more than 300 cal/mole. Other helices display half-widths of 38 cm-1 and helix-coil transition free energies of about 90 cal/mole.
The 310-helix differs from the alpha-helix in that the internal hydrogen bonding occurs between residues i and i+3 instead of i and i+4 in alpha helices.

Turn structures

The beta turn structure involves 4 amino acid residues which form a loop so that the two chain segments separated by the turn adopt an antiparallel orientation and form an i to i+3 hydrogen bond. A number of turn structures have been identified from protein structures: type I (42%,  non-helical), type II (15%, non-helical, requires Gly in position 3) and type III (18%, corresponds to one turn of 310 helix). Assignment of beta turns by means of a normal mode analysis for insulin demonstrates a strong overlapping of the different types of beta turns with the alpha-helical absorption (Krimm & Bandekar, Adv Protein Chem 1986;38:181-364). However, an absorption near 1680 cm-1 is now clearly assigned to beta turns.

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Secondary structure in proteins

The shape of the amide I band of globular proteins is characteristic of their secondary structure. With a publication by Byler & Susi (Biopolymers 1986 Mar;25(3):469-87 ) the determination of secondary structures in proteins from FTIR spectra actually started. This had become possible by the availability of high signal-to-noise ratio digitalised spectra obtained by the FTIR spectrometer and by the access to computers and software able to perform many operations on the spectra in a short time.

Deconvolution of the amide I band

The concept of Fourier self deconvolution is based on the assumption, that a spectrum of single bands (each narrow band is characteristic for a secondary structure) is broadened in the liquid or solid state. Therefore, the bands overlap and can not be distinguished in the amide envelope. A curve fitting procedure can be applied to estimate quantitatively the area of each component representing a type of secondary structure. In the pioneering work by Susi & Byler (Methods Enzymol 1986;130:290-311)  the amide I was deconvoluted with a Lorentzian line shape function and a resolution enhancement factor of 2.4 was applied. The deconvoluted spectrum was fitted with Gaussian band shapes by an iterative curve fitting procedure. The results are in good agreement with with the secondary structure information obtained from X-ray crystallographic structures of the proteins under study.

  click to enlarge


  a)     b)      
sec. structure Mean (cm-1) RMS (cm-1) Max (cm-1) Mean (cm-1) RMS (cm-1) Max (cm-1) Region (cm-1)
turns 1694 1.7 2 - - -  
  1688 1.1 2 - - -  
  1683 1.5 2 1678 2.1 5 1682-1662
  1670 1.4 2 1670 2.9 5  
  1663 2.2 4 1664 1.0 3  
alpha-helix 1654 1.5 3 1656 1.5 3  
        1648 1.6 3 1662-1645
unordered 1645 1.6 4 1641 2.0 3 1645-1637
beta sheet 1624 2.4 4 1624 2.5 5  
  1631 2.5 3 1633 2.1 4 1637-1613
  1637 1.4 3 - - -  
  1675 2.6 4 1685 2.1 4 1689-1682

a) Proteins in solution (Susi & Byler, Methods Enzymol 1986;130:290-311) or b) as hydrated film on an ATR plate (Goormaghtigh et al., Eur J Biochem 1990 Oct 24;193(2):409-20). The mean frequency of each component is reported together with the root mean square (RMS) and the maximum deviation (Max).

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Second derivative spectra and curve fitting

The spectra discussed in this chapter were collected with a IFS 66 spectrometer (Fa. BRUKER). The protein spectra are detected at a resolution of 1.5 cm-1. The native proteins were solved in water (pH 6.5). All proteins were denatured by dissolving in water and heating up to 95 oC for 50 minutes. The aqueous protein solutions to be lyophilized were frozen in liquid nitrogen. The lyophilized proteins were measured at 1.5 mg protein per 300 mg of KBr. After homogenizing the lyophilized protein and KBr were pressed into pellets by using a 12-ton hydraulic press.
The author thanks Wilfried Hartmann and Gisela Werner from the Bruker Saxonia Analytik GmbH for the access to the spectrometer facilities and for the excellent technical assistance.

The resolution-enhanced spectra allow the identification of the various secondary structures occurring in the protein. Most of the peak positions were easily found in the second derivative spectra. An example for the second derivative of a Gaussian and a Lorenztian curve is shown in this Figure. Using only the peak positions from the second derivative spectrum for lysozyme (Figure), 12 different peaks were found in the amide I region. In addition to the frequency position (a1) information on the width (a2) and the maximum absorption intensity (a0) of the individual bands can be obtained from the second derivative. However, an accurate determination of the bandwidth from the maxima in the second derivative spectrum is complicated due to the presence of neighboring peaks. Despite this inaccuracy, the obtained parameters can successfully be used as input parameters for a fitting procedure.
However, the peak positions were sometimes difficult to distinguish. This difficulty arose when either one of the two peaks showed up as a shoulder instead of a separate peak in the second derivative spectrum. In general, the bandwidths of the structural components are in the range of 8-28 cm-1. In some second derivative spectra, peaks appeared with very small band widths. In these cases it was assumed, that small fluctuations originated from noise. Using the additional parameters from the second derivative spectra for lysozyme (Figure), the resulting peak number is 6. The Table compares the secondary structure contents for different techniques with results from X-ray crystallography.
 
 

  X-ray   FTIR    
   a) STRIDE b)       a1 c)     a1  a0,a1,a2
helix 45 46 56 41 42
beta-sheet 19 17 27 29 16
turn 23 28 16 16 36
random 13 9 1 14 6

a) M. Levitt et al. J Mol Biol 1977 Aug 5;114(2):181-239
b) F. Eisenhaber et al. 1993 &1994
c) S. Luo et al. Anal Biochem 1994 Jan;216(1):67-76

   a0,a1,a2
 

Using the parameters a0, a1 and a2, the secondary structure content derived from FTIR spectra is in agreement with X-ray crystallography data. The structural components were quantified by the integrated areas of the respective peaks. This implies that the effective absorptivities can be assumed to be equal. This assumption was validatedby Byler & Susi (Biopolymers 1986 Mar;25(3):469-87).
Circular Dichroism (CD) spectroscopy is a well established technique for the analysis of secondary structure of proteins in aqueous solution. This technique seems to be less reliable for the study of aggregated proteins, inclusion bodys or membrane bound proteins due to light scattering problems associated with large membrane fragments or aggregates. FTIR spectroscopy has proved to be a powerful tool for investigations on proteins discussed above.

  LDH   FAB   CSC  
  Xray FTIR Xray FTIR Xray FTIR
helix 43 49/25 49 19/6 64 64/4
beta-sheet 19 21/15 14 39/18 1 15/14
turn 30 27/15 28 33/18 23 19/31
random 8 3/42 9 9/39 1 2/49

FTIR native/denatured (thermally denatured), all data in %

In addition to the examples decribed above we have recorded the FTIR spectra in the amide I region of LDH (lactate dehydrogenase), FAB (fab fragment, mouse antibody), CSC  (citrate synthase), LYS (lysozyme) according to the procedure described above. The second derivatives of all spectra were calculated using the spectrometer software OPUS. Before starting the fitting procedure, the obtained depths of the minima in the second derivative spectrum and, subsequently, the calculated maximum intensities were corrected for the interference of all neighboring peaks. The curve fitting is performed by stepwise iterative adjustment towards a minimum root-mean-square error of the different parameters determining the shape and position of the absorption peaks.

spectra of native proteins (click to enlarge)
LYSLDHFABCSC

The data reveal that the amide I band for all proteins consists of six or seven major components which were found in all spectra. The helix content derived from the amide I region for lysozyme, lactate dehydrogenase and citrate synthase is in agreement with the data from X-ray crystallography. The helix content in the FAB fragment of mouse antibody is too low, the beta-sheet content too high. Visual inspection of the amide I envelope of the native and thermally denatured states revealed a striking difference in the band shapes. For the native state, the band is fairly asymmetric and has a peak maximum around 1650 cm-1 which corresponds to alpha-helical structure. In contrast, the denatured proteins show an additional maximum between 1620 and 1640 cm-1, indicative of the predominance of beta-sheet and unordered structures.

spectra of thermally denatured proteins (click to enlarge)
LYSLDHFABCSC


In the following afew papers applying FTIR spectroscopy to denatured proteins and inclusion bodies are compiled

in denatured proteins
Schweers et al. J Biol Chem 1994 Sep 30;269(39):24290-7 ,
Sancho et al. Biochemistry 1995 Jan 24;34(3):1064-9 ,
Menendez et al. Eur J Biochem 1995 Dec 15;234(3):887-96 ,
Boye et al. J Dairy Res 1996 Feb;63(1):97-109 ,
Xie et al. Arch Biochem Biophys 1996 Apr 1;328(1):122-8 ,
Bramanti et al. Biopolymers 1996 May;38(5):639-53 , Biopolymers 1997 Aug;42(2):227-37 ,
Jiang et al. Biochim Biophys Acta 1996 May 23;1294(2):121-8 ,
Lee et al. Biomaterials 1996 Aug;17(16):1599-608 ,
Azuaga et al. Biochemistry 1996 Dec 17;35(50):16328-35 ,
Narhi et al. J Pept Res 1997 Oct;50(4):300-9 ,
Magdaleno et al. FEBS Lett 1997 Dec 29;420(2-3):179-85

and inclusion bodys
Chiu et al. Science 1998 Feb 20;279(5354):1190-3 ,
Tablin et al. J Cell Physiol 1996 Aug;168(2):305-13 ,
Oberg et al. Biochemistry 1994 Mar 8;33(9):2628-34.

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