Infrared Spectroscopy of Proteins

Transcription

1 Infrared Spectroscopy of Proteins Heinz Fabian 1 and Werner Mäntele 2 1 Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 2 Johann Wolfgang Goethe-University Frankfurt am Main, Frankfurt am Main, Germany 1 INTRODUCTION Infrared (IR) spectroscopy is experiencing an explosion of applications that provide molecular information in systems that range from the level of amino acids, small peptides, isolated proteins and enzymes to even more complex systems such as peptide protein complexes, membrane-bound proteins, and entire membranes. The information may be a global one such as the secondary structure composition of a protein, or it can be highly specific such as the alteration of individual bonds in an enzyme. This allows enzyme reactions induced by some experimental procedure to be followed in very high detail. Furthermore, the structural information is not restricted to a static picture but can also be obtained in real time by applying time-resolved IR techniques. An important feature is that the size of a protein or the nature of the environment does not limit the application of IR spectroscopy. We note, however, that sensitivity decreases with excessively increasing protein size. Nevertheless, investigation of proteins with molecular weight up to kda does not present a problem. Measurements can be performed in aqueous solution or organic solvents, in oriented films or deposits, as well as in detergents or membrane-like environments. Intense work over many years on the development of sample preparation techniques has led to IR sample forms which contain enough water to ensure biological integrity, but still allow IR spectroscopy even in the range of water absorbance. Thus, the argument of abiotic IR samples does not hold any more. Moreover, sample quantities have been drastically reduced from milligrams to some tens of micrograms, which makes IR spectroscopy a lot more attractive for biochemical studies. John Wiley & Sons Ltd, This article will describe instrumental techniques for steady-state absorbance and reaction-induced difference spectra. Further, sampling procedures available to obtain IR spectra of proteins, peptides, and more complex enzyme samples will be reported. This is followed by a presentation of the strategies used for band assignments, such as chemical and isotope modifications or site-directed mutagenesis, and for analysis of the spectra, such as resolution enhancement techniques or two-dimensional (2D) correlation analysis. The major part of this article will focus on illustrative examples to demonstrate the sort of information which IR techniques can provide and how this information can be extracted from the experimental data. In addition, strengths and limitations of the IR approach are discussed. This should help the reader to evaluate whether a particular system is appropriate to be studied by IR spectroscopy, and what specific advantages are gained when IR techniques are applied. The applications start with methods developed for the quantification of protein secondary structure from IR spectra. Then, examples will be presented which show how Fourier transform infrared (FT-IR) spectroscopy permits a detailed analysis of the impact of point mutations or substrate binding on the structure and stability of proteins, using peptide backbone and side-chain marker bands as conformation-sensitive monitors. Another section will deal with the problems encountered in structural studies of membrane proteins and the specific possibilities of IR techniques in this field. In this context, a number of applications of presently established reaction-induced difference techniques will be reported which illustrate the specific strengths of IR difference spectroscopy with sensitivity down to the level of individual bonds and real time observation. Furthermore, the use of isotopically labeled

2 2 Biochemical Applications compounds for the assignment, termed isotope-edited IR spectra, for the analysis of peptide protein, substrate enzyme, and peptide model membrane complexes will be discussed. The article ends with the description of the different IR spectroscopic techniques, which now permit the analysis of biochemical reactions or the monitoring of protein unfolding/folding events in real time on a picosecond to minute timescale. 2 GENERAL CONSIDERATIONS IR spectroscopy is one of the earliest experimental methods recognized as potentially useful in providing information on structural features of peptides and proteins. As early as 1950 it was demonstrated that a strong correlation exists between the position of certain bands in IR spectra of homopolypeptides and their secondary structure. 1 Later, these experimental observations were refined by making detailed vibrational analyses of the structure-sensitive amide bands in order to establish a correlation between the frequencies of these bands and the type of secondary structure, such as purely a-helical or purely b-sheet structures. 2,3 Nine such IR bands exist, which are termed amide A, amide B and amides I VII, in the order of decreasing frequency 3,4 (Table 1). Of all the amide bands, the most intense and most useful for the analysis of the secondary structure of proteins was found to be the amide I band, which represents primarily the CDO stretching vibration of the amide groups (though coupled to in-plane bending of the N H and stretching of the C N bonds) and occurs in the region cm Transmission IR spectroscopy of proteins in water Many IR experiments with proteins are carried out in aqueous solution and conducted with conventional transmission Table 1. Characteristic IR bands of the peptide linkage. Nomenclature Approximate Vibrational modes (amide) frequency (cm 1 ) A ¾3300 NH stretching in resonance B ¾3100 with 1st amide II overtone I CO stretching II NH bending and CN stretching III CN stretching and NH bending IV OCN bending, mixed with other modes V Out-of-plane NH bending VI Out-of-plane CO bending VII ¾200 Skeletal torsion geometries. IR studies of proteins in water are complicated by the fact that the H O H bending vibration of H 2 O absorbs very strongly near 1640 cm 1. A molar absorptivity of about 17 L mol 1 cm 1 appears quite low, yet it leads to an absorbance of ¾1fora10µm pathlength due to the 55 M water concentration. As a consequence, very short pathlength cells of 3 8 µm are needed for transmission measurements in the amide I region to prevent total IR absorption in the spectral regions of the water. Such short pathlengths limit the intensities of the IR bands and the signal-to-noise ratio at a given sample concentration. Consequently, relatively high sample concentrations, in the order of >10 mg ml 1 or mm, depending on the protein size, are required for the measurement. Figure 1(a) (solid line) shows the IR spectrum of the enzyme ribonuclease T1 (RNase T1) in H 2 O buffer. For these transmission measurements, a drop of 2 µl of the enzyme solution (concentration 28 mg ml 1 ) was placed between a pair of calcium fluoride (CaF 2 ) windows separated by a pathlength of 8 µm. The solvent spectrum (Figure 1a, dashed line) was measured in a matched second cell of slightly Absorbance (a) Wavenumber /cm 1 Absorbance (b) Wavenumber /cm 1 Figure 1. (a) IR spectrum of the protein RNase T1 in H 2 Obuffer (solid line) at a protein concentration of 28 mg ml 1 placed between a pair of CaF 2 windows separated by a pathlength of 8 µm, together with the buffer spectrum (dashed line) measured in a matched second cell of slightly reduced pathlength. (b) IR spectrum of RNase T1 after subtraction of the buffer spectrum.

3 Infrared Spectroscopy of Proteins 3 reduced pathlength, which takes into account the slightly lower water concentration in the protein sample measured. To obtain the spectrum of the protein, digital subtraction of solvent/buffer absorptions from the spectrum of the sample is required. For appropriate subtraction, the spectrum of the solvent/buffer should be recorded under conditions (such as temperature, ionic strength, ph, number of scans, resolution, etc.) identical with those of the sample spectrum. One reason for this is the variation of the H 2 O vibrational modes with temperature, which requires the temperature of the sample in aqueous solution and that of the reference buffer to match within 0.1 C in order to avoid artifacts caused by temperature differences. In addition to that, all other physico-chemical parameters should be matched as closely as possible for this procedure. The subtraction of water from a protein spectrum requires a reference water band that does not overlap with that of the sample. Often, a water band at around 2126 cm 1 (a combination of scissor vibration and torsional vibration) is used to ensure proper subtraction of water, which then results ideally in a straight baseline between 1900 and 2400 cm 1. If water subtraction results in derivative-type artifacts in this region, this implies that the spectroscopic characteristics of the water in the sample water are not identical with those of the reference water. The latter artifact may often be observed at very high protein concentrations (>50 mg ml 1 ), since the interaction of a protein with water causes the creation of hydration shells around the protein and therefore modifies the water vibrations and hence the shape of the water bands in the IR spectrum. Consequently, the reference spectrum of the H 2 O buffer cannot completely match the spectral conditions of the water in the protein solution (containing bulk water plus protein-bound water). The weak water band at 2126 cm 1 (which is more than six times less intense than the water band of interest at around 1645 cm 1 ) may serve as a good approximation to interactively subtract the water features. The final water subtraction, however, should be performed using a different spectral region with much stronger H 2 O absorption, such as on the slope of the O H stretching band in the vicinity of ¾3650 cm Measurements in D 2 O Experimentally it is simpler to obtain protein spectra in deuterium oxide (D 2 O, 2 H 2 O) solution than in H 2 O solution. The IR bands of D 2 O occur at lower wavenumber than those of H 2 O, creating a region of relatively low absorbance between 1400 and 1800 cm 1, an ideal window to observe the weak IR bands of the dissolved or solubilized protein. Much longer pathlengths of µm may then be used, Absorbance (a) Absorbance (b) amide II Wavenumber /cm ; amide II 1576 amide I amide I ; amide B 3295; amide A Wavenumber /cm Figure 2. (a) IR spectrum of RNase T1 in D 2 O buffer after complete H D exchange (solid line) at a protein concentration of 14 mg ml 1 placed between a pair of CaF 2 windows separated by a pathlength of 45 µm, together with the buffer spectrum (dashed line). (b) IR spectrum of fully exchanged RNase T1 after subtraction of the buffer spectrum (solid line), together with the spectrum of RNase T1 in a partially exchanged state (dashed line). hence much lower sample concentrations are required to obtain high-quality spectra. Figure 2(a) shows the IR spectrum of RNase T1 in D 2 O buffer at a protein concentration of 14 mg ml 1 (solid line) measured in an IR transmission cell with 45 µm pathlength after complete H D exchange, together with the spectrum of the D 2 O buffer (dashed line). A specific feature of protein studies in D 2 O is the exchange of protons with deuterons. Exchangeable, i.e. N-, O-, and S-bound, protons will exchange at rates which depend on the global and local accessibility of the group and on its pk a value. Consequently, the rate and extent of exchange of protons with deuterons provides a key to these parameters, and is frequently used as a tool in IR spectroscopy.

4 4 Biochemical Applications For the vibrational modes of the polypeptide backbone after solvent exchange ( 1 H 2 O ) 2 H 2 O), a substantial decrease in intensity of the amide II band (predominantly originating from peptide N H bending modes, coupled with C N stretching modes) which is centered near 1545 cm 1 in H 2 O, is observed. This band shifts down in frequency by nearly 100 cm 1 to 1450 cm 1 (amide II 0 )ind 2 O, thereby revealing side chain vibration bands of the protein such as those of arginine, tyrosine, and of aspartate and glutamate in the range cm 1. 6 At least the solvent exposed Asp and Glu residues will be deprotonated at neutral ph. The relative intensities of the amide II and the amide II 0 bands of a protein in D 2 O can by used to monitor the time course and extent of hydrogen deuterium exchange, and provide valuable information on the structure and flexibility of a protein. 7 Compared to the large effects on the amide II band, the shift of the amide I band upon deuteration of the backbone hydrogens (labeled amide I 0 by convention) is relatively small (5 10 cm 1 ). Nevertheless, individual spectral components of the amide I band reveal different exchange kinetics. Amide protons exposed to the solvent and involved in irregular or turn conformations exchange rapidly, while NH groups in stable secondary structures (a-helices and b-sheet structures) are typically resistant to exchange even after prolonged exposure to D 2 O. This greatly assists the assignment of absorption bands arising from different secondary structural classes. Despite the positive aspects, it can also complicate the interpretation of the amide I 0 region if a protein cannot be completely exchanged, because partial deuteration leads to band components with frequencies in between those of fully protonated and fully deuterated amide groups. Figure 2(b) summarizes spectral effects of partial and complete deuterium exchange on the amide bands of a protein spectrum. The dashed line in Figure 2(b) represents the IR spectrum of the protein RNase T1 that has been allowed to exchange in a D 2 O buffer for 3 h at room temperature. The residual intensity in the amide II region at about 1550 cm 1, together with the presence of the amide A band (N H stretching vibrations of the peptide groups) centered at 3295 cm 1 indicate that a significant number of the amide protons are not exchanged after 3 h of exposure to D 2 O. This situation is common in many proteins. The amide A band is the best indicator for residual nonexchanged N H groups due to the lack of other protein absorptions in the range cm 1. The same assessment cannot easily be made based on the residual intensity in the amide II region, since IR bands arising from amino acid side-chain groups overlap with the remaining amide II band, e.g. in RNase T1 the two bands are at 1515 and 1576 cm 1.Complete deuteration of RNase T1 was achieved by keeping the protein solution close to the denaturation temperature for 10 min before cooling back down to room temperature (Figure 2b, solid line). The thermal unfolding of RNase T1 is known to be fully reversible, whereas other proteins may start to irreversibly aggregate upon thermal denaturation. In most cases, keeping the temperature 10 C belowthe denaturation temperature accelerates the exchange without changing the protein structure. It is also possible to slightly destabilize the protein structure by changing the ph and/or adding chemical denaturants such as urea or, in the case of membrane proteins, increased detergent concentration, to achieve the accelerated exchange. In any case, however, it is necessary to demonstrate that this pretreatment does not irreversibly alter the native protein structure. 2.3 IR cells for transmission spectroscopy Among the IR window materials available for experiments in aqueous solution, calcium fluoride (CaF 2 )isthemost commonly because (i) it has a low refractive index which is similar to that of water, (ii) it is relatively rugged, and (iii) it is transparent from the mid-infrared (>1000 cm 1 )tothe ultraviolet (UV) region (¾190 nm) of the spectrum. Barium fluoride (BaF 2 ) has a lower spectral cutoff (at ¾800 cm 1 ) as compared to CaF 2, thus enabling additional IR spectral bands to be observed, but is significantly more soluble in aqueous solution. Materials insoluble in water (such as ZnSe, AgCl, KRS-5 or Irtran ) are available, but are characterized by a high refractive index, which results in major reflection losses and persistent interference fringes in the spectra. However, antireflection coated windows are available which partially solve this problem. Flow-through demountable cells with Luer-lock fittings, CaF 2 windows and spacers covering pathlengths from 6 to 200 µm are often used for transmission measurements. These cells are available from most IR suppliers as accessories, but have some disadvantages in their practical use. They are typically difficult to clean and to fill without accidental gas bubbles at the windows, which, if they are in the beam profile, prevent quantitative measurements. To circumvent these problems, we use custom-made IR cells of different design which consist of a flat cover disk (typically made of CaF 2 ) and a second disk of the same material, with the center deepened to form a recessed parallel surface surrounded by a trough (Figure 3a). The trough prevents direct contact of the sample with the outer part of the disk. To seal this cell, the cover disk is then pressed onto the sample disk which contains the protein solution or suspension. Simply pressing the shaped window onto the flat one is sufficient to prevent the evaporation of water for many hours at room temperature. For measurements at higher temperatures and/or for long-time experiments, the sealing surface of the disks can be covered

5 Infrared Spectroscopy of Proteins 5 (a) Cover disk Sample disk Buffer flow IR beam in (b) IR beam Sample Trough Protein/membrane sample ATR crystal (ZnSe or Ge) IR beam out Figure 3. Schematic representations of (a) a custom-made IR cell used to record spectra from samples in aqueous solution or as dispersions and of (b) an attenuated total reflection (ATR) cell for absorbance and reaction-induced difference spectroscopy. with mineral oil prior to filling and assembling the cell. This prevents both evaporation of the solvent (e.g. water) at high temperatures (e.g. 95 C), for up to several weeks, or changes of the isotope content when working with D 2 O solutions. Depending upon the diameter and the depth of the recessed surface of the window (i.e. the pathlength of the cell), only a few microliters are required to fill the cell. Moreover, this type of cells can very easily be filled with solution, assembled and disassembled, cleaned between measurements, and provides a constant pathlength which is very difficult to achieve with conventional tin or poly(tetrafluoroethylene) spacers. The pathlength of these cells can be made by manufacturers to virtually any value between a few micrometers and the maximum pathlength given by H 2 O or D 2 O absorbance. Homogeneity of the pathlength over the entire sample area, a problem for pathlengths <10 µm, can and should be controlled by comparing spectra recorded at different spots of the area with a suitable mask. 2.4 Attenuated total reflection as an alternative to transmission Another sampling technique widely used to obtain IR spectra of biological systems is ATR For ATR measurements, the sample is prepared on the surface of a trapezoidal-shaped IR-transparent crystal (Figure 3b). The IR beam is guided through the crystal in such a way that some total reflections take place at the surface. Since the IR beam provides an evanescent wave entering into the medium of lower refractive index (i.e. the region containing water, buffer, and protein), the deposition of IR absorbing matter on the crystal surface causes the IR light to be partially absorbed. The penetration depth of the IR radiation in this arrangement is strictly dependent upon the wavelength and may be up to a few micrometers. The IR spectrum thus measured contains only information on a very thin layer of the sample that is in close proximity to the surface of the crystal. This allows a spectrum of a protein in H 2 O solution to be obtained relatively easily, without much interference from IR absorption of the bulk water. Specially designed temperature-jacketed ATR cell configurations are available, which also permit experiments under temperature control. Surface adsorption, however, may significantly change the secondary structure of the protein molecules which are in direct contact with the crystal. Although the contribution of those molecules to the total absorbance measured might be small, one should proceed with caution in structural studies of water-soluble proteins by ATR spectroscopy. ATR techniques are particularly well-suited for the study of membrane-associated peptides or proteins. Here, strong adhesion of a membrane film on the ATR crystal may be of advantage. It can be forced by reconstituting membrane proteins in lipid vesicles and adhere these lipid vesicles onto the ATR crystal. This adhesion can be strong enough to allow a gentle flow of buffer over the sample surface without washing the protein away. Stable protein films can thus be obtained which allow the reversible exchange of buffers for ph titration studies or for the study of the action of effector or inhibitor molecules (see Section 11). Immobilization of protein samples on the surface of an ATR crystal typically results in a uniaxial orientation. Membrane proteins, for example, tend to orient with the membrane normal aligned with the normal of the ATR crystal. This orientation may be studied with the use of polarized radiation. Studies of immobilized samples oriented on the surface of an ATR crystal, such as proteins oriented in membranes, may be performed with polarized IR light providing information on the spatial orientations of functional groups. Compared with classical IR dichroism measurements in transmission mode, the number of layers deposited on the crystal may be much lower, usually resulting in a more uniform orientation. Furthermore, sophisticated ATR techniques allow periodic stimulation of the membrane system by modulation of an external parameter, such as temperature, concentration, electric field. This results in periodic modulation of only those absorption bands which are related to molecules, or parts of them, that are affected by the external perturbation, and even minor spectral changes within the large background of the total absorbance can be detected. 12

6 6 Biochemical Applications 2.5 Infrared microscopy The sensitivity of FT-IR spectroscopy allows a microscope to be coupled to the interferometer. The obligatory mirror optics of the microscope, which allows access to the full IR spectral range, is typically coupled to a second optical system with visible light. Objects can thus be visualized as with a conventional microscope, and spectra can be taken of the selected spots. Light throughput is consequently small as compared to the normal FT-IR instrument. Nevertheless, the reduced detector size (a consequence of the small sample spot) allows high-quality spectra to be recorded. While IR microscopy is typically used to identify and characterize structural and chemical heterogeneities in a thin sample slice, its application to photochemically unstable samples extends the use of IR difference techniques with perturbation techniques (see below). For example, studies on enzymes with photoactivated substrates ( caged compounds ) require fresh samples because of bleaching of the substrate analog. A sample with dimensions much larger than the microscope sample spot may be scanned, thus providing a fresh sample spot for every measurement. 3 DETERMINATION OF PROTEIN SECONDARY STRUCTURE Specific information on the secondary structure of proteins is obtained from the analysis of the various amide bands sensitive to the conformation. Some of these bands are more useful than others for conformational studies. By far the best characterized in this respect are the amide I/I 0 bands (see above for definition), established indicators of the protein secondary structure because of their sensitivity to hydrogen-bonding pattern, dipole dipole interaction and the geometry of the polypeptide backbone. Typically, the amide I/I 0 band of proteins consists of a series of overlapping component bands which occur as a result of the secondary structures present in such molecules. As a consequence, the individual component bands that represent different structural elements, such as a-helices, b-sheets, turns and irregular structures are often not resolved and difficult to identify in the broad amide I/I 0 band contours of the experimentally obtained spectra. Two approaches are currently used for the quantitative estimation of protein secondary structure from IR spectra: those which involve curve fitting of the amide I/I 0 band profiles and those which are based on pattern recognition algorithms. 3.1 Approaches based on curve fitting Curve fitting analysis of amide I/I 0 band profiles starts with the choice of input parameters, such as the number of component bands and their positions. Here, techniques for band narrowing such as Fourier (self) deconvolution or derivative spectroscopy are useful and essential tools. 13 Fourier deconvolution improves the degree to which the individual component bands can be resolved, with the relative integrated intensities maintained. Most commonly, the second derivative is calculated which gives a negative peak for every band or shoulder in the spectrum. Because sharp bands are enhanced at the expense of broad ones, derivatization does not preserve the integrated areas of individual components. Band narrowing techniques greatly amplify features in the spectra originating from random noise and/or uncompensated water vapor. Thus, Fourier deconvolution and derivatization should only be performed on spectra with a very high signal-to-noise ratio (preferably better than 5000 : 1) and after complete elimination of water vapor bands. 14,15 The estimated number of component bands plus their approximated width, height, and shape are then used as input parameters in an iterative least squares procedure that attempts to reproduce the measured amide I/I 0 band profile by varying these parameters. For practical reasons selfdeconvolved spectra should be subjected to curve fitting, because least squares algorithms work significantly more reliably on spectra with an enhanced profile. The fractional areas of the fitted components are taken as directly proportional to the relative quantities of structural elements they represent. The percentages of different secondary structure elements are then estimated by adding the areas of all component bands assigned to each of these structures and expressing the sum as a fraction of the total amide I/I 0 band area Critical aspects in defining typical bands for secondary structure The curve-fitting approach has some significant inherent problems. 17,18 An element of subjectivity is the assumption that the number of the component bands estimated by self-deconvolution or derivatization reflects the real number of components. In cases where bands significantly overlap, even the applied band narrowing procedures will certainly fail to separate the components present. This is especially critical in cases in which these components describe different types of secondary structure. It is therefore not surprising that it is sometimes necessary to include extra component band(s) not identified in the spectra after band narrowing, in order to achieve agreement between estimates of secondary structure from IR spectroscopy and from structural data provided by the X-ray crystallography. A very critical step is the assignment of the component bands. The assignment is normally guided by theoretical calculations and by empirical spectral-structural

7 Infrared Spectroscopy of Proteins 7 β # β # β β u Wavenumber /cm 1 Figure 4. Amide I/I 0 frequencies for protein secondary structures. Amide I (in H 2 O): dashed line frames; amide I 0 (in D 2 O): solid line frames. a: a-helices. In highly solvent-exposed a-helices the amide I 0 band can shift to cm 1 due to additional hydrogen bonding of the solvent accessible CDO groups to water. 23 The amide band in helical membrane proteins typically occurs between 1657 and 1662 cm 1, which has been suggested as indicative of more flexible and/or more stretched a-helices. 9 Calculations suggest that bands below 1640 cm 1 may also arise from the E 1 -mode motions of a-helices. 24 b: b-sheet structures. For proteins often more than one band component is observed. This reflects differences in hydrogen bonding (the stronger and shorter the hydrogen bond, the lower the frequency) as well as differences in transition dipole coupling in different b-strands. b # : b-strands in aggregated structures. IR spectra of thermally aggregated proteins are characterized by a significant increase in splitting between the low- and high-frequency b-components (b and b Ł, respectively) in comparison to that observed from b-sheets in native proteins, indicating intermolecular b-sheet structure with very strong hydrogen bonds. Similar features are found in the IR spectra of amorphous protein deposits (as in inclusion bodies) or ordered protein aggregates (amyloid fibrils) u, unordered parts of the polypeptide backbone; lp, loops; t, turns. correlations experimentally established for model polypeptides and proteins of known three-dimensional structure. The data in Figure 4 can be taken as a general guideline for the approximation of protein secondary structure based on their characteristic amide I/I 0 frequencies They illustrate that a helical and irregular structures show bands fairly close together, which sometimes causes complications in the analysis. On the other hand, amide groups in b-sheet structures of globular proteins give rise to diagnostic major bands between approximately 1622 and 1640 cm 1.Furthermore, weaker high-frequency band components are also often observed from b-sheets. Theoretical calculations suggest that it might be possible to distinguish antiparallel b-sheet structures from their parallel counterparts, since the latter lack a weak high-frequency band component arising from transition dipole coupling in antiparallel aligned b-strands. 3 Moreover, the strong low-frequency amide I 0 u α α lp lp β t t β β # β # band component of an antiparallel b-sheet is expected to absorb at slightly lower frequency when compared to that of a parallel b-sheet. Studies on model peptides experimentally support this suggestion. 21,22 Turns are associated with various bands between 1660 and 1690 cm 1, b-turns in particular with a band around 1641 cm 1. The complexity in the amide I region of a protein creates difficulties in unique assignment of an IR band component to a specific type of turn structure, although well-defined bands in IR spectra of defined turns in model structures exist. The band assignment of helices is still uncertain but two bands at around 1640 and 1662 cm 1 are under consideration. Another assumption of the curve-fitting approach is that the molar absorptivities (integrated IR intensities) of the bands associated with different secondary structural elements are identical, which is at its best a rough approximation. Studies on polypeptides and proteins have revealed that the integrated area of the amide I/I 0 absorption may vary as the structure of the polypeptide backbone is altered, indicating a change in molar absorptivities A potential difficulty is that side chains of certain amino acids also absorb in the amide I/I 0 spectral region (Table 2), which in some proteins may account for as much as 15 20% of the total integrated intensity in this region Furthermore, the contribution of amino acid side chains depends on their protonation state, which is difficult to estimate. Since their spectral contributions are not balanced in the amide I/I 0 region, a subtraction of the side-chain contributions from the experimental protein spectrum would be desirable. It has been suggested that the IR spectra of pure amino acids or simple peptides, for example tripeptides Table 2. Approximated frequencies of characteristic amino acid side chain absorption bands in the range cm 1. Side chain/assignment Absorbance maximum (cm 1 ) In H 2 O In D 2 O Asn (CDO) (n) 1678 (s) 1646 (s) Asn (NH 2 ) (υ) 1622 (m) Asp (COOH/COOD) (n) 1716 (m) 1710 (m) Asp (COO ) (n as ) 1574 (s) 1585 (s) Arg (CN 3 H C 5 ) (n as ) 1673 (s) 1605 (s) Arg (CN 3 H C 5 ) (n s ) 1633 (s) 1586 (s) Glu (COOH/COOD) (n) 1712 (m) 1706 (s) Glu (COO ) (n as ) 1560 (s) 1570 (s) Gln (CDO) (n) 1670 (s) 1635 (s) Gln (NH 2 ) (υ) 1610 (m) Lys (NH 3 ) (υ as ) 1629 (m) Lys (NH 3 ) (υ s ) 1526 (m) Phe (Ring) 1494 (w) 1498 (w) Tyr (Ring) 1614 (m) 1615 (m) Tyr (Ring) 1518 (s) 1515 (s) s, strong; m, medium; w, weak.

8 8 Biochemical Applications ALA-X-ALA, with X being the amino acid of interest, may be used for this purpose. The spectral parameters of the side chain absorption bands in the model compounds, however, provide only an approximation because the spectral features of the side chain groups in a protein are influenced by the specific microenvironment of the corresponding group. As a consequence, care should be taken upon quantitative evaluation of small amide I/I 0 components near the side chain absorbance of Asn and Gln at approximately 1675 cm 1 (n CDO) and of Arg at approximately 1675 and 1630 cm 1 (n as and n s CN 3 H C 5 ). The side chain absorbance of solvent-exposed Asp and Glu COOH modes at frequencies near 1700 cm 1 do not pose a serious problem, since they are broadened and, if at all, would only affect the high-frequency b-sheet band component (b Ł ). 3.3 Approaches based on pattern recognition Quite different approaches to estimate the secondary structure of a protein are pattern recognition methods. These methods use IR spectra of proteins with known threedimensional structures as a calibration matrix. Spectra are either used directly as a spectroscopic pattern to model the spectrum of the protein to be determined, or indirectly after reduction of the protein spectra into a number of eigenspectra which represent the most important spectral information. Mathematical tools such as partial least squares analysis 37,38 and factor analysis are normally applied for the evaluation of the protein spectra. The advantage of pattern recognition approaches is that these methods eliminate the somewhat inherent subjectivity of the band-narrowing techniques discussed above and they do not require the assignment of individual component bands to different types of secondary structure. However, these methods cannot be freely applied without considering certain limitations. For simplicity, most of the approaches involve normalization procedures that assume equal molar absorptivities for various secondary structures, quite similar to the curve-fitting analysis described previously. More importantly, the methods encounter difficulties in cases where the spectral features of the protein under study do not reflect the characteristics of the spectra within the calibration set. In such situations incorrect estimation of the secondary structure is very likely, even though the mathematical treatment of the spectral data is formally correct. In addition to these pattern recognition techniques, methods with learning capabilities such as neural nets are now emerging and these will widen the range of spectral analyses possible. 42 A very recent approach makes use of the bandshape and frequency changes resulting from controlled levels of H/D exchange to be structure-dependent, leading to an enhanced estimation of fractional secondary structure in proteins Analysis of peptide structures Some specific difficulties exist in the quantitative assessment of the secondary structure of peptides from IR spectra. Primarily, this is because the position of an IR band associated with a certain secondary structure in a peptide can be significantly different from that of the corresponding structure in a globular protein. Typically, a-helical structures in globular proteins show absorption in the range cm 1, while the position of a band of an a-helical structure in a solvent-exposed peptide may occur below 1640 cm Since the pattern recognition methods discussed previously rely on calibration spectra obtained almost exclusively from globular proteins, these approaches may not (yet) be applied for secondary structure estimation of peptides. Thus, the curve fitting approach is often applied for this purpose, despite its weakness discussed before. In proteins, differences between the H D exchange kinetics of amide protons in irregular structures (very fast) and in a-helical structures (often very slow) can aid band assignment. In peptides, however, this strategy usually does not help very much, since isotopic exchange in a-helical segments of peptides is typically also fast. While FT-IR spectroscopy has limitations in the quantification of peptide secondary structures, it represents a very useful tool for analyzing, in relative terms, structural changes by using peptide backbone and side chain bands as conformation-sensitive monitors. 45 Typical applications include the analysis of the formation of secondary structures and their changes when peptides are subjected to environmental perturbations (e.g. temperature, pressure, ph, adding of organic or membrane-mimicking solvents). Moreover, the impact of point mutations or amino acid modifications (e.g. D-amino acid substitution, amidation, phosphorylation) on the structure and stability of peptides can be analyzed. 3.5 Near-infrared spectroscopy of proteins Thus far, IR spectroscopy in the spectral range above 4000 cm 1, the near-infrared (NIR) region, has seldom been employed for structural studies of proteins. The absorptions observed in the NIR region are overtones or combinations of the fundamental stretching bands that occur in the midinfrared region. 46 The bands involved are usually due to C N, N H, and O H stretching vibrations. Generally, all these bands are much weaker than in the mid-infrared region. As a rule of thumb, overtones are at least a factor

9 Infrared Spectroscopy of Proteins 9 of ten weaker than fundamental frequencies. Moreover, the bands in the NIR region are often overlapped, making their analysis more difficult than those of the bands in the mid-infrared region. Measurements in aqueous solutions are feasible, but require relatively high protein concentrations (>30 mg ml 1 ) and cells with pathlengths of mm. Some recent examples illustrate that structural changes of proteins in aqueous solution can be successfully analyzed by using NIR spectroscopy. 47,48 4 UNFOLDING/FOLDING OF PROTEINS UNDER EQUILIBRIUM CONDITIONS 4.1 Thermal unfolding and stability The structural alterations to be studied in a protein can be induced in similar ways to those used in other methods, ranging from simple changes in temperature and pressure to extremes in ph and the addition of denaturants. For a typical pressure-dependent experiment, the protein is placed in a diamond anvil cell, and the pressure is calibrated using the quartz phonon band at around 695 cm 1 as an internal standard. 49,50 Traditionally, and by far in the majority of cases, FT-IR spectroscopy has been used to characterize unfolding processes in proteins induced by temperature. For this purpose, the protein solution in the IR cell is placed in a thermostated cell jacket inside the sample chamber of the spectrometer, and the temperature can be changed in a stepwise or continuous manner. The protein spectrum is obtained by subtracting the buffer spectrum from the spectrum of the protein solution measured at the same temperature. Heating the sample and recording the spectra can be done under computer control and thus permits an automatic scanning. 6 There are different ways to analyze the protein spectra obtained. A traditional way is the estimation of secondary structure contents by applying the different methods described before. A more appropriate approach for the characterization of temperature-induced conformational changes in proteins, at least in our view, is to construct intensity/temperature or frequency/temperature profiles for selected IR marker bands. Besides the peptide backbone amide I band components, there are several amino acid side-chain absorption bands in the range cm 1, which may serve as specific local monitors of conformational changes. Very useful is the IR band at ¾1516 cm 1, arising from the aromatic ring stretching vibration of tyrosine. The position of this band is sensitive to changes in the microenvironment of tyrosine residues, which may result from changes in both tertiary and secondary structure. 51,52 The variation of the position of this band may be smaller than a few wavenumbers, and even smaller than the spectral resolution (typically 2 or 4 cm 1 ). Nevertheless, determination of the interpolated peak wavenumber (the center of gravity) is possible to within 0.2 cm 1 and thus allows conclusions on the shift induced by unfolding/folding. Other suitable marker bands in the range cm 1 arise from COO stretching vibrations of the carboxylate moiety of the side-chain groups of aspartate and glutamate and reflect in a characteristic manner conformational changes induced by temperature 6,52 or the coordination of the sidechain COO groups to metal ions. 53 Upon thermal denaturation, many proteins start to aggregate irreversibly under conditions typical for standard FT-IR protein measurements, such as high protein concentrations and an extended time frame necessary for collection of the spectra. Here, the ratio of intensity of the b-aggregation band at cm 1 (or higher frequencies in H 2 O) to that of any of the band components of the native protein structure was found to be useful to follow thermal denaturation. 54,55 However, irreversible aggregation of proteins is not a rule but an intrinsic property for the proteins that show it. There are proteins for which thermal unfolding in the IR cell is reversible. In such cases, the intensity/temperature and frequency/temperature profiles for selected IR marker bands provide a means of determining standard thermodynamic parameters of a protein, such as transition temperatures (T m ) and enthalpy changes (1H van t Hoff). 6,51 The temperature dependence of the different IR marker bands, which probe different structural features of the protein, permits us also to address the question of whether or not unfolding can be fitted by a two-state model. Whenever the temperature profiles of two or more bands coincide, this can be considered as a strong indication in favor of such a model, although it must be remembered that corresponding signals may be insensitive to one of the transitions in some cases. If the corresponding profiles do not coincide, it clearly indicates that intermediates are present at equilibrium and hence a simple two-state transition cannot be used for analysis of the data. It is a specific advantage of the FT-IR approach that such information can be derived from a single experiment with a single sample. IR studies of the thermal unfolding of the l Cro repressor represent an illustrative example for a protein whose unfolding proceeds through a stable equilibrium intermediate. 56 In the native Cro protein, two monomeric units form a dimer by aligning the C termini of each monomer. This alignment allows the formation of an antiparallel b-ribbon across the dimer. The N-terminal parts form small globular subdomains that consist of three a-helices and a short N-terminal b-strand connected to the b-ribbon. The engineered l Cro repressor variant, in which valine-55 is replaced by cysteine

10 10 Biochemical Applications Absorbance N 20 C I 60 C tyr I 60 C U 95 C tyr 53 cm 1 β 55 cm (a) Wavenumber (cm 1 ) β α β* β* 20 (b) 1651 cm 1 (α-helices) 1624 cm 1 (β-sheet) Temperature ( C) Figure 5. (a) IR spectra (after Fourier self-deconvolution) of the disulfide-bridged Cro dimer (Cro-V55C) as a function of temperature, from the native structure (at 20 C), through the intermediate state (at 60 C), to the heat-denatured state (at 95 C). (b) Intensity vs temperature plot of the 1651 cm 1 band, reflecting the unfolding of the a-helices (upper), and of the band at 1624 cm 1, reflecting the two-step unfolding of the b-sheets (lower). [Reproduced with permission from H. Fabian, H.H. Mantsch and C.P. Schultz, Proc. Natl. Acad. Sci. USA, 96, (1999).] (Cro-V55C), spontaneously forms a disulfide cross-link between the protein subunits in the dimer. This disulfide bridge does not perturb the basic Cro fold. The IR spectrum of the native Cro-V55C protein in the D 2 O buffer after complete H D exchange and subtraction of the buffer is dominated by three bands in the amide I 0 region, which are well resolved after band narrowing by Fourier selfdeconvolution (Figure 5a). The bands at 1624 (b) and 1677 cm 1 (b Ł ) affirm the presence of an antiparallel b- sheet structure, while the band at 1651 cm 1 indicates the presence of an a-helical structure. The a-helical band disappears in the spectrum of the protein at 60 C, which now reveals a band at 1642 cm 1, typical of irregular polypeptide chains. The intensity temperature plots (Figure 5b) show that the first thermal transition involves the unfolding of the a-helices. In contrast, the characteristic b-sheet bands show only partial loss of intensity in the spectrum at 60 C, indicating that only part of the native b-sheets unfold between 20 and 60 C. It was suggested that only the short N-terminal b-strand unfolds during the first thermal transition, while the intermolecular b-sheet domain remains intact. The overall hydrogen-bonding pattern of the remaining b-sheet structure, however, is affected by the unfolding of the structure in the N-terminal region. This is indicated by the small increase in the splitting between the low and high frequency components of the antiparallel b-sheet bands (53 cm 1 at 20 C vs55cm 1 at 60 C). The b-sheets that remain in the stable intermediate start to unfold only at high temperatures above 70 C. When the thermal unfolding is complete (at 95 C), the spectrum shows a broad, featureless amide I 0 contour, typical of irregular protein structures. In the case of proteins whose thermal unfolding is reversible, the spectra of the thermally unfolded proteins provide an ideal internal standard for the normalization of the spectra of related folded proteins (e.g. wild type protein and its mutants). An essential prerequisite for this kind of data evaluation is that the IR spectra have been recorded with a very high signal-to-noise ratio, and that the stability of the instrument is also very high, otherwise minor spectral differences will be buried within the noise. In certain cases, this approach can detect minor conformational differences that are not obvious from the X-ray analysis of the corresponding proteins or could only be present in aqueous solution IR spectroscopy of proteins in the presence of chemical denaturants Obtaining IR spectra of proteins in the presence of the most commonly used denaturating agents, urea and guanidinium chloride (GdmCl) is not simple for several reasons. First, whatever denaturant that must be used at high concentrations to achieve a significant structural alteration has very strong IR bands of its own. Therefore, IR cells with very short pathlengths (5 7 µm) are required to achieve sufficient transmission in spite of the absorption of the chemical denaturant. With these cells, however, only the final mixture can be investigated, and it is not possible to add the denaturant and to follow the process. Second, the major IR bands of urea or GdmCl overlap strongly with the much weaker protein amide I band of interest and prevent their analysis. Isotopic labeling of the denaturant (e.g. using 13 CDOlabeled urea) is a suitable method which helps to circumvent this problem by shifting the urea band by ¾50 cm 1 to lower frequencies. In this way, isotopic labeling creates a clear window in the IR spectrum to observe the protein amide I bands above 1610 cm This strategy allows measurements of chemically induced unfolding/refolding transitions of proteins as described by other methods, such as UV circular dichroism (CD) spectroscopy, and enables us to compare conformational features of thermally and chemically denatured states of a protein D IR CORRELATION SPECTROSCOPY 2D IR correlation spectroscopy is a new addition to the IR-based methods employed for the enhancement of overlapping bands in the spectra and for the characterization of conformational changes in proteins. In a 2D IR correlation experiment, an external perturbation is applied

11 Infrared Spectroscopy of Proteins 11 to the sample and the IR radiation is used as a probe. The external perturbation can be any perturbation that modifies the spectrum (e.g. temperature, pressure, concentration, etc.). The correlation analysis of the spectral fluctuations leads to 2D maps that increase the spectral resolution by spreading peaks along a second dimension and that reveal the order of the actual sequence of events induced by the perturbation. In general, two types of spectral representations are obtained, synchronous and asynchronous plots. 60,61 The synchronous 2D correlation plot recognizes the similarity between the variations of spectral intensities to a perturbation, whereas the asynchronous 2D correlation plot detects differences between the variations of spectral intensities. The synchronous 2D plot is characterized by autopeaks located on the diagonal and by cross-peaks that are placed off-diagonal. Autopeaks are observed when spectral features vary as a function of the applied perturbation. Cross-peaks can either be positive or negative. They reflect correlated changes that occur simultaneously in the same (positive) or in the opposite direction (negative). The asynchronous 2D plot is characterized by missing autopeaks and asymmetric cross-peaks with positive and negative intensities. Asynchronous spectra are of particular interest, because they allow one to distinguish spectral intensities that vary slightly out of phase (i.e. delayed or accelerated). Thus, the 2D IR approach provides a sensitive means of detecting a sequence of events in response to changes induced by external perturbing factors in a protein. The 2D IR methodology has been used to analyze temperaturedependent spectra of peptides and polypeptides, 62,63 to improve the resolution of individual band components in the IR spectra of myoglobin, 64 or to detect sudden changes in the hydration of ovalbumin that precede the unfolding of the protein. 65 An illustrative example is the 2D IR correlation analysis of the thermal unfolding of the l Cro-V55C repressor protein. 66 Figure 6 shows the synchronous and asynchronous 2D IR correlation plots demonstrating the Figure 6. 2D correlation analysis of the IR spectra of the l Cro-V55C repressor protein measured between 20 and 60 C (A, synchronous; B, asynchronous plot). The correlation plots are represented as 2D color intensity maps (left) and also as pseudo-3d maps (right) with the following color coding: blue, set to zero in the synchronous plots; green, set to zero in the asynchronous plots (see color bars). b and b Ł, the low- and high-frequency components, respectively, of the antiparallel b-sheet band; b1 andb2, two low-frequency components; a, a-helices.). [Reproduced with permission from H. Fabian, H.H. Mantsch and C.P. Schultz, Proc. Natl. Acad. Sci. USA, 96, (1999).]