Ultraviolet Resonance Raman Study of Drug Binding in DihydrofolateReductase, Gyrase, and Catechol O-MethyltransferaseVincent W. Couling,* Peer Fischer,* David Klenerman,* and Walter Huber#*Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, England, and#F. Hoffman-La Roche, 4002 Basel, SwitzerlandABSTRACT This paper presents a study of the use of ultraviolet resonance Raman (UVRR) spectroscopic methods as ameans of elucidating aspects of drug-protein interactions. Some of the RR vibrational bands of the aromatic amino acidstyrosine and tryptophan are sensitive to the microenvironment, and the use of UV excitation radiation allows selectiveenhancement of the spectral features of the aromatic amino acids, enabling observation specifically of their change inmicroenvironment upon drug binding. The three drug-protein systems investigated in this study are dihydrofolate reductasewith its inhibitor trimethoprim, gyrase with novobiocin, and catechol O-methyltransferase with dinitrocatechol. It is demon-strated that UVRR spectroscopy has adequate sensitivity to be a useful means of detecting drug-protein interactions in thosesystems for which the electronic absorption of the aromatic amino acids changes because of hydrogen bonding and/orpossible dipole-dipole and dipole-polarizability interactions with the ligand.INTRODUCTIONA key process in the development of new drugs is elucida-tion of the nature of the interaction between the drug mol-ecule and the target protein. Such knowledge then makes itpossible to make systematic structural modifications of thedrug molecule to optimize the interaction. There are avariety of techniques currently available for obtaining in-formation about drug-protein interactions, such as the mea-surement of kinetics and binding affinities. The two meth-ods that have traditionally been employed to obtainstructural information of proteins and drug-protein com-plexes are x-ray diffraction and NMR spectroscopy. Both ofthese methods have disadvantages: x-ray diffraction re-quires the preparation of a crystal, which can be timeconsuming or even impossible; and NMR spectroscopy isnot easily applied to larger proteins of more than a fewhundred amino acids.Other analytical techniques that can be applied to proteinsin solution are circular dichroism, ultraviolet absorption,and fluorescence spectroscopy, but these all have limita-tions. Circular dichroism is useful mainly in probing theglobal structure of proteins; however, far UVCD can yieldinformation about protein secondary structure and nearUVCD can probe the local environment of aromatic resi-dues, especially tryptophan (Woody and Dunker, 1996).Although ultraviolet absorption spectroscopy is widely em-ployed to study the phenolic hydrogen bonding (H-bonding)of tyrosine (Tyr, Y) (Cantor and Schimmel, 1980; Dem-chenko, 1983), this approach requires one to differentiatethe effect of Tyr H-bonding from the contribution due to allof the other amino acids (Hildebrandt et al., 1988). Withfluorescence spectroscopy, if any tryptophan (Trp, W)amino acids are present, they will tend to dominate thespectrum, the signals from other residues often being com-pletely swamped. Furthermore, it is difficult to quantita-tively relate changes in fluorescence yields to structuralchanges in proteins.Resonance Raman (RR) spectroscopy with ultraviolet(UV) excitation radiation has been demonstrated to be avaluable new method for the study of biological molecules(Spiro, 1987; Asher, 1993a,b). Recent advances in UVlasers (Asher et al., 1993), especially the extension of lasersources into the deep UV region, have led to the establish-ment of UVRR spectroscopy as an active area of research.This is evidenced by recent papers in the literature, whichdocument the use of UVRR in the study of the structures ofhemoglobin (Copeland et al., 1985), cytochrome c (Cope-land and Spiro, 1985), insulin (Rava and Spiro, 1985a),lactalbumin (Kronman et al., 1981), angiotension (Cho andAsher, 1996), enkephalins (Takeuchi et al., 1992), humanserum albumin (including its ligand-binding modes) (Hashi-moto et al., 1995), the extracellular domain of the humantumor necrosis factor receptor (Tuma et al., 1995), as wellas a variety of DNA structures (Takeuchi and Sasamon,1995). In this paper we explore the feasibility of usingUVRR spectroscopic methods to help elucidate aspects ofdrug-protein interactions.The advantages of UVRR as compared to normal Ramanspectroscopy are much improved selectivity and sensitivity.Both advantages arise because of the large enhancement ofthe cross section of Raman scattering that occurs when theexcitation laser frequency closely matches the electronicabsorption frequency of a particular chromophore. The in-creased sensitivity of UVRR means that spectra can beobtained at lower sample concentrations, typically on theorder of 1 mg ml21, which obviates the problem of proteinaggregation, which can occur in solutions at higher concen-Received for publication 18 February 1998 and in final form 21 April 1998.Address reprint requests to Dr. D. Klenerman, Department of Chemistry,University of Cambridge, Lensfield Road, Cambridge CB2 1EW, England.Tel.: 44-1223-336481; Fax: 44-1223-336362; E-mail: [email protected]. Couling’s permanent address is Department of Physics, University ofNatal, Pietermaritzburg 3200, South Africa.© 1998 by the Biophysical Society0006-3495/98/08/1097/10 $2.001097Biophysical Journal Volume 75 August 1998 1097–1106trations. The increased selectivity means that only the res-onantly enhanced chromophores will be featured, yieldinggreatly simplified spectra of proteins. The extent and selec-tivity of the enhancement depend on the excitation wave-length used, and this can be exploited to good effect, be-cause in the UV range it is the aromatic amino acids of aprotein, namely Tyr, Trp, and phenylalanine (Phe, F), whichare enhanced, and not the aliphatic amino acids. As the vastmajority of amino acids in proteins are generally aliphatic,the efficacy of using UV excitation radiation is immediatelyapparent.It is well known that some of the vibrational bands of Tyrand Trp are sensitive to the microenvironment. Indeed,detailed information about the microenvironment of thesearomatic side chains can be obtained from, for example,variations in intensity of the vibrational modes, or changesin the intensity ratio of Fermi resonance doublets. A specificexample is the Fermi resonance doublet of Tyr, which