Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2007.00320.x |
Fourier Transform Infrared Spectroscopic
Analysis of Protein Secondary Structures
Jilie KONG and Shaoning YU*
Department
of Chemistry, Fudan University, Shanghai 200433, China
Received: March 15,
2007
Accepted: April 29,
2007
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 20745001 and 20525519), the National Basic Research Program of China
(2007CB914304), and the Educational Foundation of Fudan University (EYH1615002)
*
Corresponding author: Tel, 86-21-55664974; Fax, 86-21-65641740; E-mail,
[email protected]
Abstract Infrared spectroscopy is one of the oldest and well
established experimental techniques for the analysis of secondary structure of
polypeptides and proteins. It is convenient, non-destructive, requires less
sample preparation, and can be used under a wide variety of conditions. This
review introduces the recent developments in Fourier transform infrared (FTIR)
spectroscopy technique and its applications to protein structural studies. The
experimental skills, data analysis, and correlations between the FTIR
spectroscopic bands and protein secondary structure components are discussed.
The applications of FTIR to the secondary structure analysis, conformational
changes, structural dynamics and stability studies of proteins are also
discussed.
Keywords FTIR; protein structure; protein dynamic
Infrared (IR) spectroscopy is one of the oldest and well established
experimental techniques for the analysis of secondary structure of polypeptides
and proteins [1–5]. The use of stable and powerful laser has led to the development
of the Fourier transform (FT) method for IR data acquisition and reliable
digital subtraction. The availability of modern computers has enabled the rapid
and powerful FTIR data processing and conversion. FTIR spectroscopy is
recognized as a valuable tool for the examination of protein conformation in H2O-based solution, as well as in deuterated forms and dried states,
resulting in a greatly expanded use in studies of protein secondary structure
and protein dynamics in the past decade [4–17]. Although X-ray
crystallography provides the most detailed information concerning positions of
individual atoms in the protein structure, it is not, however, possible for all
proteins to form a quality crystal for such analysis. In addition, the
crystallographic data of a protein cannot be easily extrapolated to the dynamic
properties of the proteins in solutions. Nuclear magnetic resonance
spectroscopy can be an alternative to X-ray crystallography in solution, but
the interpretation of nuclear magnetic resonance spectra of a large protein is
a very cumbersome process [18]. Thus, the vibrational spectroscopies, such as
FTIR and circular dichroism (CD), are still important and commonly used
techniques for protein structure and dynamics studies.
FTIR spectroscopy is a measurement of wavelength and intensity of the
absorption of IR radiation by a sample. The IR spectral data of high polymers
are usually interpreted in terms of the vibrations of a structural repeat unit
[2,3,19]. The polypeptide and protein repeat units give rise to nine
characteristic IR absorption bands, namely, amide A, B, and I–VII. Of these,
the amide I and II bands are the two most prominent vibrational bands of the
protein backbone [3–5]. The most sensitive spectral region to the protein secondary
structural components is the amide I band (1700–1600 cm–1), which is due almost entirely to the C=O stretch vibrations of the
peptide linkages (approximately 80%). The frequencies of the amide I band
components are found to be correlated closely to the each secondary structural
element of the proteins. The amide II band, in contrast, derives mainly from
in-plane NH bending (40–60% of the potential energy) and from the CN stretching vibration
(18–40%)
[3], showing much less protein conformational sensitivity than its amide I
counterpart [3]. Other amide vibrational bands are very complex depending on
the details of the force field, the nature of side chains and hydrogen bonding,
which therefore are of little practical use in the protein conformational
studies. Banker described the nine amide vibration modes and some standard
conformations in detail in his review [20]. The characteristic IR bands of the
proteins and peptides are listed in Table 1.
In 1980’s, the practical use of FTIR was severely limited by factors
such as low sensitivity of the instrument, interfering absorption from aqueous
solvent, lack of understanding of the correlations between specific backbone
folding types and individual component bands. It was considered back then that
the IR spectrum was difficult, if not impossible, to be used in an aqueous
solution unless deuterium oxide was used as a solvent [7], because water
absorbs strongly in the most important spectral region at approximately 1640 cm–1. Even in D2O solution, usually only
qualitative information was obtained because the components of absorption bands
associated with specific substructures, such as a-helix and b-sheet, can not
be resolved. Later, as more protein structures were solved by X-ray
crystallography, and the computational procedures for the resolution
enhancement of broad IR bands of protein were developed, the IR spectra of
polypeptides and proteins in D2O solution could be assigned [4–7]. Later the
amide I band assignments for proteins in H2O
solution were also made [8–11]. The computerized FTIR instrumentation has improved the
signal-to-noise ratio and allowed extensive data manipulation. The new
band-narrowing methods, Fourier self-deconvolution (FSD) and second derivative,
have not only enriched the qualitative interpretation of the IR spectra, but
also provided a basis for the quantitative estimation of protein secondary
structure [17]. Now, for most globular proteins, people can acquire IR spectra
quantitatively under certain conditions and use the FTIR technique to study the
structure and function of proteins and peptides [11–17].
The singular advantages of FTIR over other techniques are that
spectra can be obtained for proteins in a wide range of environments, requiring
less time and sample, and direct correlations between the IR amide I band
frequencies and the secondary structure components can be found. The goal of
this paper is to review the recent developments in FTIR technique and its
applications to protein secondary structure analysis and conformational study.
Bypassing the basic theory of molecule vibration, we will focus on the
experimental skills, data analysis, IR band assignment and applications to
protein structural study. In this work, all spectra presented were measured by
BOMEM FTIR spectrophotometer (ABB Bomen Inc., St-Laurent, Canada) and Beckman
FH-01 IR cell (Beckman Coulter Inc., Fullerton, USA) with CaF2 windows. The cell path-length is 7.5 mm for H2O and 50 mm for D2O solution. BOMEM GRAMS/32 software (ABB Bomen
Inc.) was used for data acquisition and analysis.
Protein FTIR Data Analysis and
Band Assignment
Data analysis
High sensitivity to small variations in molecular geometry and
hydrogen bonding patterns makes the amide I band uniquely useful for the
analysis of protein secondary structural composition and conformational changes
[4,11]. In the amide I region (1700–1600 cm–1), each type of secondary structure gives rise to a somewhat
different C=O stretching frequency due to unique molecular geometry and hydrogen
bonding pattern. However, other than the distinctive absorbance maxima, the
observed amide I bands of proteins are usually featureless, due to the
extensive overlap of the broad underlying component bands, which lie in close
proximity to one another and are instrumentally unresolvable. Thus,
mathematical methods such as resolution-enhancement technique are necessary to
resolve the individual band component corresponding to specific secondary
structure. Fig. 1(A) is the IR spectrum of cAMP receptor protein (CRP)
in buffer. The individual underlying components can not be visualized without
resolution enhancement.
Mathematical data analysis methods can be used to “enhance”
the resolution of the protein spectrum, allowing the intrinsically broad
components to be narrowed and separated beyond the instrument resolution
[1,22]. The mathematical band-narrowing process does not actually increase the
instrumental resolution, but rather increases the degree of separation by
narrowing the half-bandwidth of individual components for easier visualization.
This band-narrowing process is achieved at the expense of spectral quality of
the original band, which leads to a degradation of signal-to-noise ratio.
Several methods have been developed to estimate quantitatively the relative
contributions of different types of secondary structures in proteins from their
IR amide I spectra in solution, including FSD-curve fitting [7,22], second
derivative analysis [4,7,11], partial least-squares analysis [23], and data
basis analysis [24]. The FSD-curve fitting and second derivative analysis are
the two most popularly used methods.
The theoretical backgrounds of IR data handling techniques,
including FSD and second derivative, have been discussed in detail by Susi and Byler
[4,6]. The FSD method was also reviewed by Surewicz and Mantsch [18]. The key
meaningfulness of the FSD method is to select the conditions that achieve the
maximum band narrowing while keeping the increase in noise and the appearance
of side-lobes at minimum [6,14,22]. This method is based on the assumption that
a spectrum of single bands (each narrow band is characteristic of a secondary
structure) is broadened in the liquid or solid state. Therefore, the bands
overlap and can not be distinguished within the amide envelope. An alternative
approach to “enhance” the resolution of overlapping IR bands is based
on the generation of nth order derivative band files. This can be
carried out in the frequency (wavenumber) domain of the spectrum. Second
derivative spectra allow the identification of various secondary structures
present in the protein [6]. Most of the peak positions are easily found in the
second derivative spectra. An improved method for carrying out second
derivative analysis has established the utility of the method for obtaining
quantitative as well as qualitative determination of a-helix, b-sheet, random
and turn structures [11]. A curve fitting procedure can be applied to calculate
quantitatively the area of each component representing a type of secondary
structure [4,8–11]. Fig. 1(B) shows the second derivative spectrum of CRP,
which was obtained according to the methods of Dong et al. [11]. Both
FSD and second derivative methods were included in BOMEM GRAMS/32 software.
Second derivative analysis was used in this work. Manipulation procedures were
carried out in accordance with Dong et al. [11].
The overlap of secondary structural components is significant in the
amide I region, even after mathematical resolution enhancement. Some
investigators considered that the valley between two adjacent peaks of equal
intensity must be 20% lower than the peak tops that could be resolved [1,25].
Band assignment
Quantitative analysis of protein secondary structure is based on the
assumption that protein can be considered as a linear sum of a few fundamental
secondary structural elements. Comparisons of IR spectra with high-resolution
X-ray crystal structures of proteins could establish necessary
spectra-structure correlations. Over the years, many correlations between IR
spectra and particular protein structure have been established. The amide I
band components can be assigned by studying their frequency behavior in which
the protein secondary structure is known by other techniques. As mentioned
above, the amide I band (17001600 cm–1) is due
mainly to the C=O stretching vibration (approximately 80%) of the amide groups
coupled with little in-plane NH bending (<20%) [3]. The extract frequency of
this vibration band depends on the nature of hydrogen bonding involving the C=O
and NH moieties [3]. In turn, this is determined by the secondary structure
adopted by the polypeptide chain, reflecting the backbone conformation and
hydrogen-bonding pattern. Strictly speaking, the observed amide I band contours
of proteins or polypeptides consist of overlapping component bands,
representing a-helices, b-sheets, turns and random structures. It is needed to establish a
correspondence between IR spectra and the various types of protein secondary
structure. Assignments of the amide I band component to each secondary
structure element are available for proteins in both D2O and H2O media [4–16].
In D2O solution, it has been revealed that the
broad protein amide I band contours can be decomposed into a number of
components. A component centered between approximately 1658 and 1650 cm–1 has been assigned to the a-helix, which is consistent with both
theoretical calculation [3] and the observation of bands in the spectra of a-helical
proteins [4,26]. Bands near 1663 cm–1 are
assigned to 310 helices [3,4,11], although this
structure is rarely found in proteins. More than one b-component has been
observed in the spectra of many b-sheet proteins. Bands in the regions of 1640–1620 cm–1 and 1695–1690 cm–1 have been assigned to b-sheet by many
authors [3,4,23]. Theoretical calculation of b-sheets also predicts an IR
active mode between approximately 1695 and 1670 cm–1 [3]. These b-components are often complicated by the presence of more than one
band above 1670 cm–1. The assignment of bands around
1670, 1683, 1688 and 1694 cm–1 to b-turns has been
proposed [3]. Turns are also associated with a characteristic band around 1665
cm–1. The unordered conformation (usually referred to as random coil) is
usually associated with the IR band between 1640 and 1648 cm–1 [3]. The distinguishing feature of random coil is that it is
non-repetitive. Such assignment is supported by the position of a prominent
band in the spectra of apparently order-less proteins [7].
H2O as a solvent is much more preferable than D2O for studying protein structure [8–11]. D2O changes the protein properties somewhat in comparison with the
native ones. In H2O solution, the bands between 1654 and 1658 cm–1 are assigned to a-helix, which is supported by human hemoglobin A and bovine
Myoglobin proteins, and are expected for proteins with a-helical structure [11].
The bands between 1642 and1624 cm–1 are
assigned to b-sheets components through the IR spectra of immunoglobulin G and
concanavalin A, which contain more than 60–70% b-sheet
structures and almost no a-helix. In globular protein, it has been observed that approximately
30% of amino acid residues reside in b-turn conformations [11,28]. The bands located
at 1688, 1680, 1672, and 1666 cm–1 are assigned
to b-turn
structures. The characteristic band for random coil conformation can be
assigned to the band located at 16482 cm–1.
Some minor or rare structures might interfere with the band
assignments discussed above. For example, the b-turn band at approximately
1665 cm-1 is near the characteristic IR band representing 310-helices. Vibrations of some amino acid side chains might make small
contributions to the intensity of characteristic protein amide bands. In
addition, the experimental procedure might also bring spectral error. All these
complications indicate that there is no simple correlation between the IR
spectra and secondary structural components. Caution has to be exercised in the
interpretation of IR spectra of proteins. Table 2 shows the deconvoluted
amide I band frequencies and assignments to secondary structure for proteins in
D2O and H2O.
Subtraction of background
The accuracy of subtraction of large H2O bands
is always a concern. H2O has strong IR absorbance with
three prominent bands around 3400 (O-H stretching), 2125 (water association),
and 1645 cm–1 (H-O-H bending). The amide I
vibration for proteins absorbs between 1600 and 1700 cm–1, overlapping directly with the H2O
bending vibrational band at 1645 cm–1. The
intensity of the water absorbance at 1645 cm–1 is approximately an order of magnitude higher than the amide I
absorbance of proteins. For IR study of protein in H2O
solution, water absorption in the 1600–1700 cm–1 region might be the biggest problem. It is much easier if the
spectroscopic study is carried out in D2O
solution because there is no absorption spectrum of D2O in the
region where the amide I and II bands are observed. A large path-length (i.e.
50 mm)
IR cell can be used and results in a higher signal-to-noise ratio, a factor of
particular importance for proteins of low solubility. However, the amide I band
frequencies are strongly affected by the H-D exchanges in the peptide linkages
[4,7]. The effect of these exchanges on protein structural properties is not
fully understood, especially under incomplete H-D exchange conditions.
Furthermore, because the exchange of D for H can affect the strength and length
of hydrogen bonds, it is possible that protein secondary structures might be
altered by the replacement of H2O by D2O.
Therefore, H2O-based media have the advantage of providing a more native
environment.
The greater sensitivity of the FTIR instrument and new H2O subtraction program make it possible to obtain a good protein FTIR
spectrum in H2O solution [23,29,30]. The problem of water
absorption can also be limited by using an IR cell of sufficiently small
path-length (6–10 mm) to permit IR radiation passing through the material under
observation. By using the same cell to record both the reference spectrum and
the spectrum of protein solution under identical scan conditions, the aqueous
water contribution can be removed from the spectrum of the protein solution by
digital subtraction. Furthermore, progress in the development of methods for
spectral data analysis makes it easier to distinguish the individual components
within the intrinsically overlapped amide I band contours [11,12].
Nevertheless, the accurate measurement of frequency and intensity of the amide
bands is elusive [12]. The short cell path-length makes it more difficult to
match the path-lengths of sample and reference cells [12,31]. Typically, the
same cell was used to record the spectra of both sample and reference, although
the IR cell drying and reloading steps might slightly alter the cell
path-length. In order to get a successful subtraction of absorption bands due
to liquid water and gaseous water in the atmosphere, one must have a criterion
for determining whether or not the absorption by water is correctly
compensated, that is, the region of the spectrum where no absorption by the
sample but absorption by water is present [31]. Two criteria have been
established to judge whether the spectrum is good or not [12]. First, the bands
originating from water vapor must be subtracted accurately from the protein
spectrum between 1800 and 1500 cm–1. Second,
a straight baseline must be obtained from 2000 to 1750 cm–1. Many investigators have used a straight baseline between 2000 and
1750 cm–1 as the standard for judging the
success of water subtraction to obtain protein spectra [12,32,33]. By using two
criteria, the average experimental errors of the spectra at the amide I and II
band maxima could be less than 3% and 1.5%, respectively [8].
Empirically, in order to obtain a high quality protein spectrum,
high protein concentration (>10 mg/ml in H2O) [34] and small cell
path-length (6–10 mm) are needed. The identical scan conditions are used to record
buffer reference spectrum and the spectrum of protein solution. In addition,
water vapor, as well as water (or buffer) itself, must be subtracted accurately
from the protein spectrum. As mentioned above, using a straight base line
between 2000 and 1750 cm–1 as the
standard to judge the successfulness of water subtraction would lead to a
higher quality of protein spectra.
Application of FTIR to Protein
Secondary Structure
FTIR spectroscopy has been
used to study the secondary structure composition, structural dynamics,
conformational changes (effects of ligand binding, temperature, pH and
pressure), structural stability and aggregation of proteins.
Estimation of protein
secondary structure
Proteins are frequently
referred to as having a certain fraction of structural components (a-helix, b-sheet,
etc.). The secondary structural composition is some of the most important
information for a structure-unknown protein. Therefore estimation of protein
secondary structure is one of the major applications of the FTIR technique.
The quantitative estimation of protein secondary structure is based
on the assumption that any protein can be considered as a linear sum of a few
fundamental secondary structural elements, and the percentage of each element
is only related to the spectral intensity (the molar absorptivity of C=O
stretching vibration for each secondary structural element is essentially
same). To analyze the amide I band component, FSD or second derivative spectra
need to be curve fitted. The areas of FSD or second derivative spectra correspond
to different types of secondary structure components. It is considered that the
accuracy of measured band areas in the second derivative amide I spectra
depends upon the correct positioning of the baseline [11]. For the globular
protein, the correlation coefficient between IR and X-ray estimates of a-helices, b-sheets, b-turns and
remainder was 0.98, 0.99, 0.90 and 0.92, respectively [8]. Dong and colleagues
reported the distribution of secondary structures determined from the amide I
spectra of globular proteins in aqueous solutions, which was nearly identical
to the amount computed from crystallographic data [12].
Fig. 2 shows the analysis procedure of
second derivative FTIR spectra of CRP. The percentage of a-helices was 41,
calculated by the relative area at 1653 cm–1. The percentage of b-sheets was 36, calculated by the relative area near 1637 cm–1. The percentage of b-turns was 18, calculated by adding the areas of all b-turn bands
between 1670 and 1690 cm–1. The
band area at 1648 cm–1 was assigned to random coil.
This data is consistent with the X-ray crystallography data. Table 3
lists some results calculated from the FTIR method compared with the X-ray
crystallographic method, including the data of CRP and pyruvate kinase. From
these data, the results from FTIR and X-ray crystallography methods match well.
Kinetics of H-D exchange
related to conformation
Conformation and structural dynamics are essential to proteins for
carrying out their proper biological function. The structural dynamics of a
given conformation is likely to influence the activity of the protein. H-D
exchange has been used extensively in studies of structural dynamics of
proteins [36–38]. It has been established that the rates at which the amide
proton exchange with solvent deuterium reflect the structural dynamics of
proteins [39] and they are sensitive to the secondary structural composition
and experimental conditions such as pH, temperature and pressure [36–39]. At constant
experimental conditions, a more rapid rate of exchange implies a greater
flexibility and motion in the structural region of the exchange.
FTIR spectroscopy has been used in connection with H-D exchange in
polypeptides and proteins. The intensity changes of the amide I and II bands
and the intensity change of the predominant secondary structural elements (a-helix and b-sheet) can be
determined. It is suggested that it is more convenient to base H-D exchange
investigations on apparent intensity changes of the amide II band [4], because
it was not adversely interfered by the absorption bands of H2O, HOD, or D2O. Barksdale and Rosenburg
suggested that the protein amide H-D exchange ratio could be presented as the
fraction of unexchanged amide proton [40]. A calculation equation was
established and used by some investigators [16,41]. Fig. 3 shows the H-D
exchange ratio of pyruvate kinase in Tris buffer as a function of time,
indicated by both original and second derivative FTIR spectra.
Most IR structural dynamics studies are focused on the H-D exchange
of proteins on a global scale [16,36–38]. One may also focus on the exchange rate
of secondary structure components. As a successful example, Dong et al.
focused on the exchange rate of CRP secondary structural components, and
obtained some useful information. CRP is a dimer with two functional domains in
each subunit. There is a bias in the distribution of secondary structural
elements between the two functional domains, namely extensive b-sheets in the
cAMP binding domain and predominant a-helices in the DNA binding domain [42,43].
They took advantage of this feature and facilitated the interpretation of the
ligand-induced conformational and structural dynamics changes [16].
Protein stability
Generally, protein stability studies can provide information on the
folding/unfolding and structural stability of protein molecules. The advantage
of FTIR amide I spectroscopy over other techniques is that the IR method can,
in principle, monitor the folding/unfolding process of all parts of the protein
simultaneously [44]. However, FTIR amide I spectroscopy has not been widely
used as a method of choice in studies of protein folding. The reason is that
obtaining good IR spectra of proteins in the presence of chemical denaturant,
which must be used at high concentration to ensure protein unfolding, is not
simple and has had only limited success [15]. First, IR cell with short
path-lengths is required for preventing a saturation of IR detector by the
absorption of chemical denaturant (Hg/Te/Cd detector saturates near 4.0 M
GdnHCl at the cell path-length used), which currently prevents application of
this method to proteins that are fairly resistant to GdnHCl denaturation
[15,45]. Second, the major IR bands of denaturant (urea or GdnHCl) mask weaker
amide I bands. Previous works on GdnHCl-induced denaturation of proteins have
shown that accurate subtraction of GdnHCl absorption is difficult above 1660 cm–1 [46]. 13C urea in H2O shifts
the urea IR absorption to 1624 cm–1, leading
to a similar problem. To circumvent this, one might resort to 13C-labeled urea in D2O, which shifts the C=O band of
urea from 1618 cm–1 to 1562 cm–1 [44,45], leaving a clear window in the IR spectrum for observing
the protein amide I band above 1600 cm–1. The
combination of IR cells with a short path-length and 13C-labeled
urea allows us to measure the IR spectra of protein in the presence of high
concentrations of chemical denaturant and gain useful information from the
conformation-sensitive amide I band of protein [44,45].
The use of a wide range concentration of denaturant GdnHCl in FTIR
spectroscopy has been reported. Bowler and colleagues successfully monitored
GdnHCl-induced cytochrome c denature by FTIR spectroscopy [15]. The
first point to be considered regarding the spectra of denatured proteins in the
presence of GdnHCl is the reliability of the subtraction procedure.
Protein-bound water molecules do not seem to have an altered vibrational
spectrum from unbound water molecules [8,9]. The effects of over- and
under-subtraction are best evaluated after resolution enhancement by second
derivative methods [15]. It is apparent that the major effects of inaccurate
subtraction are limited to the wavelength region between 1690 and 1665 cm–1 [15]. One important criterion is subtraction of GdnHCl must be done
so as to produce a smooth amide I band shape with no discontinuities and to
maintain the amide I to amide II peak intensity ratio close to that observed
for the native protein. Improper subtraction is evidenced by a doubled peak in
the amide I region or an amide I peak with a concave discontinuity [15]. When
the amount of amide I and GdnHCl intensity becomes comparable during the
subtraction, the spectrum shows two peaks, allowing the GdnHCl intensity to be
removed with a good degree of certainty. Although one cannot be certain of the
absolute accuracy of the denaturant subtraction, by using consistent methods a
high degree of precision in subtraction can be achieved allowing comparison of
differences in the spectra of denatured states of closely related proteins,
even in the region between 1690 and 1665 cm–1 [15]. The instrument saturates near 4.0 M GdnHCl at the cell
path-length used, which is another limitation that cannot be neglected. The
highest GdnHCl concentration that is actually used should be less than 3.5 M by
FTIR spectroscopy. Fig. 4 shows the subtraction of GdnHCl. In the
figure, A represents the absorbance spectrum of CRP with 0.8 M GdnHCl, B
represents the absorbance spectrum of 0.8 M GdnHCl, and C represents the
absorbance spectrum of CRP after proper subtraction. Compared with Fig. 1(A),
this IR absorbance spectrum is smooth and continuous with a normal intensity
ratio of the amide I over the amide II band, so the subtraction of 0.8 M GdnHCl
is acceptable.
Unlike chemical induced unfolding, thermal induced protein unfolding
followed by FTIR spectroscopy can avoid limitations in saturation and
subtraction. This technique has been widely used in protein aggregation studies
[46,47]. Current theoretical and experimental evidence for protein aggregation
suggests that aggregates are formed from partially folded intermediates [48].
Aggregation of proteins is a problem with serious medical implications and
economic importance. To develop strategies for preventing protein aggregation,
the mechanism and pathways by which protein aggregate must be characterized. In
contrast to far-ultraviolet CD, IR spectroscopy is insensitive to light
scattering, thus providing a valuable tool for protein aggregation studies. The
thermally-induced aggregation processes of the majority of proteins studied by
FTIR can be described with a two-state model, the predominant secondary
structural element (a-helix or b-sheet) decreases as a function of temperature and is concomitantly
replaced by intermolecular b-sheet aggregates [47]. As a successful example, Dong et al.
adopted FTIR to investigate protein aggregation by a combination of thermal and
chemical denaturation, thus providing a means to populate and characterize
aggregation intermediates. They suggested that this method is valuable for
studying the aggregation processes of a wide range of proteins. The
identification and characterization of aggregation intermediates might lead to
new interdiction strategies for amyloidogenic human disease, as well as
improvements in industrial processing, storage, and delivery of therapeutic
proteins.
Secondary Structures of Proteins
Adsorbed onto Aluminum Hydroxide
Recently, a new IR technique was developed for studying protein
structure at low concentration in solution [50]. Adsorbing proteins onto
alhydrogel provides a means of obtaining FTIR spectra to study secondary structure
and conformational changes of proteins in aqueous solution at very low
concentration. This new procedure effectively lowers the concentration
requirement for FTIR studies of proteins in aqueous solutions by at least
40-fold, as compared to the conventional FTIR method. This technique permits
FTIR study of proteins to be carried out in the same concentration range as
those used for CD and fluorescence, making it possible to compare structural
information obtained by three commonly used techniques in the biophysical
characterization of proteins.
Complexity of FTIR Spectrum:
Side-chain Absorption
The FTIR studies of peptides and proteins have made efforts in
identifying characteristic frequencies and determining their relations to the
structures of protein molecules. The data analysis depended on empirical
correlations of the spectra of chemically similar molecules, and occasionally
yielded significant insights into the dependence of the spectrum on the
conformation of the polypeptide chains [3]. It is very important to recognize
that there is no simple correlation between IR spectra and the secondary
structure elements of proteins. FTIR spectra are complicated in that every
single spectrum has its own characteristics due to different micro-circumstances.
Different ambient circumstances, including side-chain absorbance, make it
difficult to precisely assign the secondary structure and frequency. The band
assignment shown in Table 2 is only empirical. The estimation of secondary
structure with the FTIR method should be considered only as a good
approximation [8].
The main difficulty in a quantitative study of the protein spectra
is the estimation of side-chain absorption, which must be taken into account in
the analysis of protein spectra. The contribution of side-chain in the globular
protein spectra is 10–30 % of the overall absorption [8]. This absorption is superimposed
on the peptide absorption and can somewhat change the amide I band spectral
parameters. Some amino acid residues, especially arginine, asparagine,
glutamine, aspartic and glutamic acids, lysine, tyrosine, histidine and
phenylalanine have intensive absorption in the amide spectral region [8,49].
Venyaminov and Kalnin [8] and Chirgadze et al. [49] have established the
absorption parameters of these residues in the amide spectral region in H2O solution (Table 4). The quantitative estimation of amino
acid side-chain groups permits more refined analysis of the secondary structure
of polypeptides and proteins by FTIR, allows the protein spectra to be
investigated in detail and achieves a better interpretation of the observed
spectral effects. This should be kept in mind when analyzing protein spectra,
especially when the content of these residues is high [8].
Summary
FTIR spectroscopy is a well-established experimental technique for
studying the secondary structural composition and structural dynamics of
proteins. Armed with mathematic resolution enhancement techniques, various
methods of FTIR data analysis have been well developed. The correlations
between IR spectra and protein secondary structures have been established. The
amide I band component assignments to protein secondary structure elements,
such as a-helix, b-sheet, b-turn and random structures, are available for proteins in H2O as well as D2O media. One could adopt those
techniques to study various protein systems for different purposes. Sample
backgrounds have to be subtracted accurately from the protein spectrum with
strict procedure(s). To obtain high-quality IR spectra, relatively high
concentrations of proteins (e.g. >10 mg/ml) and small cell path-length (6–10 mm) are needed
for proteins in H2O solution unless protein was adsorbed onto
alhydrogel.
The singular advantage of FTIR over other techniques is convenience.
IR spectra can be obtained for proteins in a wide range of environments with a
small amount of sample. Other than estimating the content of protein secondary
structures, IR can also provide information on protein structural stability and
dynamics. The FTIR spectrum is also complex, and some characteristic bands of
secondary structure elements might overlap. The background subtraction
procedure could also bring experimental error. Estimation of side-chain
absorption must be taken into account in the analysis of protein spectra. All
the complications indicate that caution has to be exercised in the
interpretation of IR spectra of proteins. When estimating the percentage
contents of protein secondary structures, a combination of FTIR and CD is
recommended for increasing prediction accuracy.
Acknowledgements
The authors appreciate Dr. James C. LEE at the University of Texas
Medical Branch (Texas, USA) for suggesting writing this review and Dr. Aichun
DONG at the University of Northern Colorado (Colorado, USA) for critical review
of the manuscript.
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