Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

5-methyl thienylacrylic acid resonance Raman Principally through the efforts of crystallographers, we are being presented with an ever expanding atomic view of the biological world. Although this brings into focus many questions regarding the mysteries of function, techniques are needed that facilitate the transition in our understanding from structure to function. Raman spectroscopy is one of these; because the Raman effect involves an intimate interplay between atomic positions, electron distribution, and intermolecular forces, it sits at the bridgehead between structure and function. Thus, the Raman technique can answer questions that lie at the heart of issues such as ligand macromolecule recognition and enzymatic catalysis. Raman spectroscopy involves analyzing the scattered photons from a laser beam focused into the sample solution (1Carey P.R. Biochemical Applications of Raman and Resonance Raman Spectroscopies. Academic Press, New York1982Google Scholar). The inelastic scattered photons (the Raman spectrum) provide information on molecular vibrations that, in turn, yield data on molecular conformation and environment. At its most effective, Raman spectroscopy can provide exquisite detail from an important site in a much larger macromolecular complex. Although Raman was first applied to the definition of biological molecules in the 1930s (2Edsall J.T. J. Chem. Phys. 1936; 4: 1-6Crossref Scopus (41) Google Scholar), the giant has remained drowsy due the difficulties both in obtaining high quality data and in interpreting those data. Considerable advances have been made in these areas in the past few years, and the giant is stirring! A major goal of this review is to provide biochemists with enough information to determine whether the Raman technique could provide structural insights into their systems. The specific issues addressed are which type of systems are amenable to study and what information could be obtained. Practically, present-day sample requirements are for 20 μl of clear solution, where the target molecule is in the 100–300 μm range. Because the number of vibrational modes of a molecule is 3 n − 6, where n is the number of atoms, the complete Raman spectrum of a macromolecule is exceedingly complex. Thus, Raman is most suited to systems where it is possible to focus upon a small region of interest, e.g. a ligand-receptor or enzyme-substrate binding site. Historically, this condition was achieved by using resonance Raman spectroscopy (1Carey P.R. Biochemical Applications of Raman and Resonance Raman Spectroscopies. Academic Press, New York1982Google Scholar) to obtain the intensity-enhanced spectra from chromophores at specific sites in macromolecules. Recent technical advances mean that similar information can now be gleaned from non-chromophoric systems, markedly broadening the application of the technique. The information obtained can be very detailed, exceeding the level of resolution found in x-ray or NMR analyses (3Carey P.R. Tonge P.J. Acc. Chem. Res. 1995; 28: 8-13Crossref Scopus (48) Google Scholar, 4Wang J.H. Xiao D.G. Deng H. Webb M.R. Callender R. Biochemistry. 1998; 37: 11106-11116Crossref PubMed Scopus (42) Google Scholar, 5Deng H. Wang J. Callender R.H. Grammer J.C. Yount R.G. Biochemistry. 1998; 37: 10972-10979Crossref PubMed Scopus (18) Google Scholar). In addition to providing structural data, the Raman spectrum can also reveal changes in the distribution of electrons in a bound ligand and details of active site-ligand interactions, such as hydrogen bonding strengths. Raman spectroscopy is beginning to fulfill its potential to contribute to structural biology because the three roadblocks that impeded its application to biological systems have been all but removed. These were low sensitivity, interference from fluorescence background, and problems with data interpretation. Sensitivity has increased several orders of magnitude with the corresponding decrease in concentration requirements because of advances in optical filters and photon detectors (6Kim M. Owen H. Carey P.R. Appl. Spectrosc. 1993; 47: 1780-1783Crossref Scopus (40) Google Scholar). Fluorescence interference is now minimized by using deep-red excitation in the 650–800 nm range, made possible by the advent of photon detectors with high efficiency in this region (7Dong J. Dinakarpandian D. Carey P.R. Appl. Spectrosc. 1998; 52: 1117-1122Google Scholar). Problems with interpreting Raman spectra have receded with the availability of “friendly” software packages (8Frisch M.J. Pople J.A. et al.Gaussian 94. Gaussian Inc., Pittsburgh, PA1995Google Scholar) and ever increasing computational power that enable us to calculate, ab initio, the Raman spectra of mid-sized molecules (of the size of many ligands or co-factors found at biological sites). Interpretation is further strengthened by a comparison of the calculated and experimental shifts in Raman peak positions when a molecule is substituted with stable isotopes. Recently, this approach has been used to characterize hydrogen bonding in a complex of adenosine deaminase with a transition state analogue (9Deng H. Kurtz L.C. Rudolf F.B. Callender R. Biochemistry. 1998; 37: 4968-4976Crossref PubMed Scopus (15) Google Scholar) and to discriminate between different protonation states of dihydrofolate binding to dihydrofolate reductase (10Deng H. Callender R. J. Am. Chem. Soc. 1998; 120: 7730-7737Crossref Scopus (32) Google Scholar). The Raman spectrum of 5-methyl thienylacrylic acid (5-MTA)1 is shown in Fig.1; the 5-MTA entity has been used extensively as a probe of protease active sites (3Carey P.R. Tonge P.J. Acc. Chem. Res. 1995; 28: 8-13Crossref Scopus (48) Google Scholar, 11Doran J.D. Carey P.R. Biochemistry. 1996; 35: 12495-12502Crossref PubMed Scopus (35) Google Scholar, 12Dinakarpandian D. Shenoy B. Pusztai-Carey M. Malcolm B.A. Carey P.R. Biochemistry. 1997; 36: 4943-4948Crossref PubMed Scopus (13) Google Scholar). The spectrum was obtained by focusing a laser beam into a solution in methanol and by analyzing the scattered light 90° to the direction of the beam using a Raman spectrometer. A small percentage of the scattered photons exchange energy with the vibrational energy levels (or, crudely, the “vibrations”) of the molecules in solution. Thus, by analyzing the scattered photons information on the vibrational motions of atoms in molecules is obtained. These motions are a function of molecular conformation, of the distribution of electrons in the chemical bonds, and of the molecular environment. Thus, interpretation of the Raman spectrum provides information on all these factors. This is the underlying principle behind using Raman spectroscopy for defining the detailed chemistry of molecules at biological sites (1Carey P.R. Biochemical Applications of Raman and Resonance Raman Spectroscopies. Academic Press, New York1982Google Scholar). With present day computational power, commercially available software packages allow us to undertake high level quantum mechanical calculations on molecules such as 5-MTA acid and to predict the stable conformational states for this molecule as well as the infrared and Raman active vibrations (13Dinakarpandian D. Carey P.R. Biospectroscopy. 1999; 5 (in press)Crossref PubMed Scopus (1) Google Scholar). Such calculations put interpretation of the data on a sure footing. They also reveal the complex nature of vibrational spectra; many peaks are due to vibrational motions that include contributions from many atoms in the molecule. However, some vibrations are more or less localized in molecular groupings and three such are indicated in Fig. 1 (the C=O and C=C stretching vibrations and the breathing-type motion of the thienyl ring). We will see below how the carbonyl peak can provide detailed chemical information on the chemistry of this group in serine protease active sites. In addition, the C=C stretch and ring modes can be used to follow the redistribution of π-electrons for 5-MTA in cysteine protease active sites (11Doran J.D. Carey P.R. Biochemistry. 1996; 35: 12495-12502Crossref PubMed Scopus (35) Google Scholar), and marker bands in the 1000–1200 cm−1 region give the conformation, cis or trans, about the =C–C=O single bond (13Dinakarpandian D. Carey P.R. Biospectroscopy. 1999; 5 (in press)Crossref PubMed Scopus (1) Google Scholar, 14O'Connor M.J. Dunlap R.B. Odom J.D. Hilvert D. Pusztai-Carey M. Shenoy B. Carey P.R. J. Am. Chem. Soc. 1996; 118: 239-240Crossref Scopus (14) Google Scholar). The absorption spectrum of the 5-MTA chromophore shows a maximum in the near UV at 324 nm. If a laser wavelength far from this electronic transition, e.g. near 650 nm, is used to generate the Raman spectrum the resulting spectrum is relatively weak. If, however, we use an excitation wavelength near 330 nm the coincidence between this and the absorption band leads to large (103 or more) intensity enhancement. This is the resonance Raman (RR) effect, and because of its high intensity it has been the method most used for obtaining vibrational spectra at biological sites. RR plays a key role in obtaining data from natural chromophoric sites such as occur in heme (15Kitagawa T. Mizutani Y. Coordination Chemistry Rev. 1994; 135/136: 685-735Crossref Scopus (102) Google Scholar, 16Blackwood M.E. Rush III, T.S. Romesberg F. Schultz P.G. Spiro T.G. Biochemistry. 1998; 37: 779-782Crossref PubMed Scopus (68) Google Scholar, 17Hu S. Smith K.M. Spiro T.G. J. Am. Chem. Soc. 1996; 118: 12638-12646Crossref Scopus (471) Google Scholar) and metalloproteins (18Dong S. Spiro T.G. J. Am. Chem. Soc. 1998; 120: 10434-10440Crossref Scopus (31) Google Scholar). Moreover, time-resolved RR is a powerful means of following changes at a chromophoric site in a rapidly evolving system such as cytochrome oxidase (19Kitagawa T. Ogura T. Prog. Inorg. Chem. 1997; 45: 431-479Crossref Scopus (2) Google Scholar) or a peptide in the early stages of folding (20Lednev I.K. Karnoup A.S. Sparrow M.C. Asher S.A. J. Am. Chem. Soc. 1999; 121: 4076-4077Crossref Scopus (57) Google Scholar). However, for stable or slowly evolving systems, increases in sensitivity now allow us to obtain Raman difference spectra from specific sites under non-resonance conditions. The RR approach remains the only method available to probe short-lived species (with half-lives of less than a second down to the picosecond range), and RR studies on some reactive acyl enzymes exemplify the detailed structural information that can be obtained. In the late 1970s and 1980s α,β-unsaturated acyl enzymes of the form R–C=C–C(=O)–O-chymotrypsin (one of these acyl groups is 5-MTA, seen in Fig. 1) were good candidates for RR analysis (3Carey P.R. Tonge P.J. Acc. Chem. Res. 1995; 28: 8-13Crossref Scopus (48) Google Scholar, 21MacClement B.A. Carriere R.G. Phelps D.J. Carey P.R. Biochemistry. 1981; 20: 3438-3447Crossref PubMed Scopus (31) Google Scholar, 22Phelps D.J. Schneider H. Carey P.R. Biochemistry. 1981; 20: 3447-3454Crossref PubMed Scopus (12) Google Scholar, 23Carey P.R. Phelps D.J. Can. J. Chem. 1983; 61: 2590-2595Crossref Google Scholar). These acyl enzymes are models for the natural acyl enzymes formed during peptide hydrolysis. The former have absorption maxima near 350 nm, and using near UV laser sources, RR spectra could be generated of the acyl groups in the active sites. Moreover, it was possible to obtain spectral data from the unstable acyl enzymes, prior to deacylation, at high pH in a rapid mixing rapid flow system. The focus of the work was the RR feature due to the acyl's C=O group, which could be used to monitor this group prior to nucleophilic attack in the active site. Three findings emerged from these studies. 1) As pH was varied, changes in the C=O stretch region occurred with the same p Ka as that for the deacylation kinetics leading to the conclusion that the p Ka of neighboring His-57 was being probed (24Tonge P.J. Carey P.R. Biochemistry. 1989; 28: 6701-6709Crossref PubMed Scopus (23) Google Scholar). 2) At high pH, a linear relationship was found between the position of the C=O stretch and the log of the deacylation rate constant (25Tonge P.J. Carey P.R. Biochemistry. 1990; 29: 10723-10727Crossref PubMed Scopus (50) Google Scholar). It extends over a change in rate constant of 17,000-fold. Moreover, an empirical relationship between C=O frequency and bond length could be used to follow C=O bond length changes of the order of 0.001–0.01 Å. This approach relies on setting up accurate structure-spectra correlations on a series of “small” model compounds (in this case generated by IR and x-ray studies on crystals of cyclic and heterocyclic organic compounds (26Horvath G. Illenyi J. Pusztay L. Simon K. Acta Chim. Hung. 1987; 124: 819-822Google Scholar)) and then using these to interpret the changes seen in the (resonance) Raman spectra in terms of exquisitely accurate structural definition. 3) Shifts in the C=O stretch were postulated to be because of changes in the active site –C=O hydrogen bonding strengths, and this effect, too, may be quantitated. By undertaking H-bonding studies, e.g. involving the ester of the compound seen in Fig. 1 in CCl4, with a number of hydrogen bond donors, it is possible to derive a relationship between the shift in the C=O stretch and the strength of the hydrogen bond(s) to it. Across the present series of acyl enzymes, the enthalpy of hydrogen bonding changes by 57 kJ mol−1(27Tonge P.J. Carey P.R. Biochemistry. 1992; 31: 9122-9125Crossref PubMed Scopus (64) Google Scholar, 28Tonge P.J. Fausto R. Carey P.R. J. Mol. Struct. 1996; 379: 135-142Crossref Scopus (37) Google Scholar). The work discussed above on acyl serine proteases utilized near UV lasers operating near 350 nm to generate RR spectra of the bound acyl groups. The spectra were recorded using double or triple monochromators (to separate the “Raman” from the interfering “Rayleigh” photons) and detected by a single photomultiplier or, later, an optical multichannel analyzer. Encouraged by the relationship cited above, attempts were made to extend the studies to acyl cysteine proteases, e.g. R–C=C–C(=O)–S–papain, and to other α,β-unsaturated thiol esters such as hexadienoyl-CoA binding to enoyl-CoA hydratase (29Tonge P.J. Fausto R. M. Pusztai-Carey M. Carey P.R. Biospectroscopy. 1995; Scopus Google Scholar). these acyl groups have absorption near 350 nm and are candidates for RR studies. However, spectra could be obtained from these systems because α,β-unsaturated thiol esters are and the and in the laser beam used to generate the RR data. to the a of optical filters to the photons and high quantum efficiency photon detectors increased the sensitivity of Raman (6Kim M. Owen H. Carey P.R. Appl. Spectrosc. 1993; 47: 1780-1783Crossref Scopus (40) Google Scholar). In terms this that it was to use the resonance high quality Raman data were using excitation near or 650 nm. This is far from absorption and the are by the laser beam P.J. Carey P.R. Callender R. Deng H. J. Am. Chem. Soc. 1993; Scopus Google Scholar). the Raman spectrum of the bound Raman difference spectroscopy was where the spectrum of the is from the spectrum of the complex R. Deng H. Rev. Struct. 1994; PubMed Scopus Google Scholar, M. Carey P.R. J. Am. Chem. Soc. 1993; Scopus Google Scholar). The difference spectrum due to the bound ligand with the of some modes are conformational changes upon ligand such as the 5-MTA seen in Fig. 1 are Raman give to relatively Raman under non-resonance because of the and system. this the ligand modes the difference ligands that less in the difference spectrum in both the and and these are a of information on changes in the interpretation of many of these is in its J. Carey P.R. J. Am. Chem. Soc. 1997; Google Scholar, J. B.A. Carey P.R. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). The Raman difference spectra for acyl cysteine proteases, by absorption spectral data, different insights from those for the serine (11Doran J.D. Carey P.R. Biochemistry. 1996; 35: 12495-12502Crossref PubMed Scopus (35) Google Scholar, J.D. Tonge P.J. Carey P.R. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). these in the cysteine protease active sites are that about a major of the π-electrons in the acyl at the active site is one of the important of the acyl and it was that the with its the acyl to in the transition state (11Doran J.D. Carey P.R. Biochemistry. 1996; 35: 12495-12502Crossref PubMed Scopus (35) Google Scholar). on active electron have been further by Raman difference studies on acyl enzymes involving the enzymes, thiol and D. Shenoy Hilvert D. D. M. Carey P.R. Biochemistry. 1999; PubMed Scopus Google Scholar). The Raman data for these 5-MTA acyl enzymes that the acyl group in the active sites. However, when the active sites are e.g. in by by a a conformational state is and this second is Thus, the acyl group is to from a region of to one where into the structure of with studies (13Dinakarpandian D. Carey P.R. Biospectroscopy. 1999; 5 (in press)Crossref PubMed Scopus (1) Google Scholar, D. Shenoy Hilvert D. D. M. Carey P.R. Biochemistry. 1999; PubMed Scopus Google Scholar), it was possible to provide a molecular for the and of the acyl groups by In the in the active by the the acyl group to about its =C–C=O single it from an where it to one where is a near the thienyl ring and at the carbonyl because of hydrogen and an Thus, to be achieved by a of electron and as shown in the in Fig. The above on the use of the 5-MTA group to many of the details on structure and chemistry that can be from Raman data. This molecule was one of the chromophoric acyl groups as RR of active sites in early studies. It is of a natural for the enzymes however, with the of Raman and the use of or excitation to fluorescence we are to a of systems. such involves the that the as shown in attempts to undertake RR studies of were by the of the thiol However, using a Raman difference in (6Kim M. Owen H. Carey P.R. Appl. Spectrosc. 1993; 47: 1780-1783Crossref Scopus (40) Google Scholar), Raman difference data for the complex were obtained K. J. D. Tonge P.J. J. Carey P.R. Biochemistry. 1995; PubMed Scopus Google Scholar). These that the active site of can about a complete of the electrons in turn, into how the chemical of the on the ring by an is The for this in the active site of R. G. H. G. D. Biochemistry. 1996; 35: PubMed Scopus Google Scholar) are very similar to those found for the cysteine proteases and shown in Fig. At the C=O is electron by and an with electron by an near the with the high Raman studies on were by concentration The were to 1 and were by the of solution during concentration However, in we were to a to its for (7Dong J. Dinakarpandian D. Carey P.R. Appl. Spectrosc. 1998; 52: 1117-1122Google Scholar). With this concentration requirements were to 100–300 data for a rate of and high quality spectra were obtained for several with different and of the J. H. L. D. Carey P.R. Biochemistry. 1999; PubMed Scopus Google Scholar). of these the of evolving with H. J. Carey P.R. D. Biochemistry. 1999; PubMed Scopus Google Scholar). It the binding to where the group been to The was to be and a series of changes were detected over a of several Fig. Raman difference spectra recorded and the to the The data for spectrum was only but the spectral of several species can be (in the the peaks at and cm−1 that is the of the bound to the active site of the However, this rapidly with is also at early for peaks at and and detailed analysis J. H. L. D. Carey P.R. Biochemistry. 1999; PubMed Scopus Google Scholar) shows that these are because of the form of the bound to the 5 the spectra and the is that of in the active site of the The Raman and absorption data provide the following for the spectral the a of the about by of the The the of which in its its form to the large of Thus, early in the are of and binding to the However, the the of to give the Thus, in a few all the is to and all the is to the and we see the of changes to the In these studies 1) that Raman difference spectra were to species in a complex evolving and to in the and 2) that species (with of of or could be at the Although are where the Raman approach provides important information on large macromolecular such as L. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, L. Biochemistry. 1999; PubMed Scopus Google Scholar, Rev. Struct. 1999; 28: PubMed Scopus Google Scholar) it may most use in defining small of large In that it techniques such as x-ray that provide the is that, are that have high that are to This is an of active the of the is The target molecules most suited for Raman difference spectroscopy are those that have relatively Raman which means that have systems because these are and give to Raman systems such as are less amenable for Raman with the above the are good that Raman spectroscopy will an increasing in structural In the of interest, involving the few have seen the Raman technique from being to a very few systems to being a means of questions for a of It is now possible to follow changes in chemistry in have been for Raman because of their However, using or laser high quality non-resonance Raman data can now be obtained from the ring system of M. Carey P.R. J. Am. Chem. Soc. 1993; Scopus Google Scholar), and these to reveal a of are as few studies involving these in the near the potential for Raman to molecular information for a of ligands binding to a or target has to be In Raman us to obtain the Raman spectra of under J. M. D.J. T. 1990; PubMed Scopus Google Scholar). quality data can be obtained for and has the potential to provide detailed information on in the as well as the and can provide a between the of the and solution studies. in and computational power are leading to models of macromolecular binding sites 1997; PubMed Scopus Google Scholar, J. Chem. 1998; PubMed Scopus Google Scholar), and Raman may have a role in providing which to the of will Raman spectroscopy an important in many as for now it will be as but it may an for in the chemistry of many of small molecule The is in its the of is are to and the are to interpret in a to and for contributions to the work discussed in this review and to for some of their most