HIV-1 Tat Peptide Binding to TAR RNA by Electrospray Ionization

Kristin A. Sannes-Lowery,† Peifeng Hu,‡ David P. Mack, Houng-Yau Mei, and Joseph A. Loo*. Chemistry Department, Parke-Davis Pharmaceutical Researc...
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Anal. Chem. 1997, 69, 5130-5135

HIV-1 Tat Peptide Binding to TAR RNA by Electrospray Ionization Mass Spectrometry Kristin A. Sannes-Lowery,† Peifeng Hu,‡ David P. Mack, Houng-Yau Mei, and Joseph A. Loo*

Chemistry Department, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent complexes formed from the interaction between HIV-1 Tat peptide and Tat protein with TAR RNA. Both positive ion and negative ion ESI mass spectra showed a maximum stoichiometry of 3:1 between Tat peptide and TAR RNA. However, the higher order complexes only occurred at high relative concentrations of Tat peptide. The 1:1 Tat peptide-TAR RNA complex is believed to involve only specific interactions, whereas the higher order complexes involve nonspecific interactions. Relative binding affinities between Tat peptide and TAR RNA and its various mutants (TAR missing the three-nucleotide bulge, TAR with a poly(ethylene glycol) linker in the bulge region, and TAR with a poly(ethylene glycol) linker in the loop region) can be differentiated by competitive binding experiments and ESI-MS measurements. The gas phase mass spectrometry experiments are consistent with solution phase studies, as they show that mutations in the bulge region reduce TAR RNA affinity to Tat peptide. Protein recognition of ribonucleic acids (RNA) is an important area of study for many biochemical systems.1 In particular, human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by the binding of viral regulatory proteins to specific RNA target sequences. Tat protein from HIV is a viral transactivator that is essential for viral replication.2 Tat is required to increase the rate of transcription from the HIV long terminal repeat (LTR), and its action is dependent on the region near the start of transcription in the viral LTR called the transactivation responsive (TAR) element, which is located at the 5′-end of mRNA.3 The basis of protein-RNA recognition, in general, is just beginning to be understood.1 Much has been learned about RNA-protein interactions, for example, from studies on the RNA binding of bacteriophage R17 coat protein.4 More and more protein-RNA complex structures have been determined in recent

years.5-7 The first example of an NMR structure of a proteinRNA complex was provided by Varani and co-workers for the 22 kDa complex between the human U1A protein (RNA binding domain) and the polyadenylation regulatory element; both RNA and protein undergo conformational changes upon complexation.8 Developing new methodologies for studying protein-RNA interactions would be useful for providing additional insight on their structures. To characterize the noncovalent interactions between proteins and their nucleic acid substrates, mass spectrometry (MS) with matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have been applied. Biological compounds with Mr greater than 150 kDa can be measured with subpicomole sensitivity by ESI-MS and MALDI-MS. The MALDI process does not typically allow for survival of an intact, noncovalently bound complex, although more recent studies suggest that such complexes may be observed by MALDI-MS.9-11 Positive ion MALDIMS was used to study the complexation between proteins and small oligonucleotides.9 However, the use of covalent crosslinking between interacting macromolecules is a more viable and generally applicable method currently for MALDI-MS analysis. Barofsky and co-workers have reported on the MS characterization of UV cross-linked protein-nucleic acid complexes.12,13 The stoichiometry of the binding partners is readily assigned from the mass measurement. Furthermore, the sites of interaction for protein-DNA complexes can be localized by proteolysis of the cross-linked complex. Mass analysis of the proteolytic peptides still bound to DNA defines the DNA-binding domain of the native protein. A similar study was recently reported by Cohen et al., in which the solution phase structure of the transcription factor DNA-binding protein, Max, was characterized.14 However, the noncovalent complex was studied; the results from the limited proteolysis of the protein in the absence and in the presence of DNA were evaluated to provide information about cleavage site accessibility. Protein regions that are buried or are involved in

* Address correspondence and reprint requests to this author. Phone: (313) 996-7515. Fax: (313) 998-2716. E-mail: [email protected]. † Current address: Wacker Silicones Corp., 3301 Sutton Rd., Adrian, MI 492219397. ‡ Current address: Baxter Health Care Corp., Route 120 & Wilson Road, WG 3-15, Round Lake, IL 60073. (1) RNA-Protein Interactions; Nagai, K., Mattaj, I. W., Eds.; Frontiers in Molecular Biology Series; Oxford University Press: Oxford, 1994. (2) Frankel, A. D. Curr. Opin. Gen. Develop. 1992, 2, 293-298. (3) Dingwall, C.; Ernberg, I.; Gait, M. J.; Green, S. M.; Heaphy, S.; Karn, J.; Lowe, A. D.; Singh, M.; Skinner, M. A. EMBO J. 1990, 9, 4145-4153. (4) Romaniuk, P. J.; Lowary, P.; Wu, H. N.; Stormo, G.; Uhlenbeck, O. C. Biochemistry 1987, 26, 1563-1568. (5) Draper, D. E. Annu. Rev. Biochem. 1995, 64, 593-620. (6) Aboul-ela, F.; Karn, J.; Varani, G. J. Mol. Biol. 1995, 253, 313-332.

(7) Ramos, A.; Gubser, C. C.; Varani, G. Curr. Opin. Struct. Biol. 1997, 7, 317323. (8) Allain, F. H.-T.; Gubser, C. C.; Howe, P. W. A.; Nagai, K.; Neuhaus, D.; Varani, G. Nature 1996, 380, 646-650. (9) Tang, X.; Callahan, J. H.; Zhou, P.; Vertes, A. Anal. Chem. 1995, 67, 45424548. (10) Lecchi, P.; Pannell, L. K. J. Am. Soc. Mass Spectrom. 1995, 6, 972-975. (11) Rosinke, B.; Strupat, K.; Hillenkamp, F.; Rosenbusch, J.; Dencher, N.; Kruger, U.; Galla, H. J. J. Mass Spectrom. 1995, 30, 1462-1468. (12) Jensen, O. N.; Barofsky, D. F.; Young, M. C.; von Hippel, P. H.; Swenson, S.; Seifried, S. E. Rapid Commun. Mass Spectrom. 1993, 7, 496-501. (13) Jensen, O. N.; Kulkarni, S.; Aldrich, J. V.; Barofsky, D. F. Nucleic Acids Res. 1996, 24, 3866-3872. (14) Cohen, S. L.; Ferre-D’Amare, A. R.; Burley, S. K.; Chait, B. T. Protein Sci. 1995, 4, 1088-1099.

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protein-protein or protein-DNA interactions should be protected from proteolysis. ESI-MS has shown greater utility for studying molecular interactions largely composed of noncovalent forces. Molecular ions (albeit multiply charged) for intact peptide, protein, and oligonucleotide species are generated by ESI with little, if any, fragmentation. Through collisional activation methods induced in the vacuum regions of the ESI mass spectrometer (e.g., ESI interface, collision cell),15 dissociation of the multiply charged molecules can occur for even large biomolecules,16,17 thereby generating structurally informative product ions. It is this ability to control the level of molecular activation that has led practitioners of ESI-MS to investigate its applicability for studying weakly bound biochemical complexes. The possible advantages of mass spectrometry for characterizing noncovalent complexes include the speed and sensitivity of the analysis and the fact that the stoichiometry of the binding partners is provided by the mass accuracy of MS.18 The gentleness of electrospray ionization has allowed mass spectrometry to directly detect several examples of noncovalently bound complexes.18,19 Complexes between peptides and proteins,20 protein-cofactor,21,22 enzyme-receptor,23 and other biochemical complexes24 can remain intact in the gas phase for the mass spectrometer to measure. The stoichiometry of the binding partners can be deduced easily by accurate mass measurement of the ligands and the intact complex. The observation of noncovalent protein-DNA complexes by ESI-MS was recently reported. Complexes between the DNA binding domain of PU.1 and specific double-stranded DNA25 and gene V protein with single-stranded DNA26 were studied by Smith and co-workers. The stoichiometry of the noncovalent association between various oligonucleotides with serum albumin protein was measured by ESI-MS.27 Moreover, the binding constants were calculated from MS titration experiments and Scatchard analysis and were found to be in reasonable agreement with those measured by capillary electrophoresis. We have examined the binding of Tat protein and peptides derived from Tat to TAR RNA and to mutant analogs of TAR RNA by ESI mass spectrometry. Experimental conditions were established to allow both positive and negative ion detection of the noncovalently bound complexes. By comparing the ESI-MS results to data from solution phase methods, our results indicate that ESI mass spectra of protein-RNA complexes reflect the specific interactions found in solution. (15) Smith, R. D.; Loo, J. A.; Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R. J. Am. Soc. Mass Spectrom. 1990, 1, 53-65. (16) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991, 63, 24882499. (17) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993, 65, 425-438. (18) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23. (19) Loo, J. A. Bioconjugate Chem. 1995, 6, 644-665. (20) Ogorzalek Loo, R. R.; Goodlett, D. R.; Smith, R. D.; Loo, J. A. J. Am. Chem. Soc. 1993, 115, 4391-4392. (21) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1991, 113, 8534-8535. (22) Drummond, J. T.; Ogorzalek Loo, R. R.; Matthews, R. G. Biochemistry 1993, 32, 9282-9289. (23) Ganem, B.; Li, Y.-T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 78187819. (24) Ganem, B.; Li, Y.-T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 62946296. (25) Cheng, X.; Morin, P. E.; Harms, A. C.; Bruce, J. E.; Ben-David, Y.; Smith, R. D. Anal. Biochem. 1996, 239, 35-40. (26) Cheng, X.; Harms, A. C.; Goudreau, P. N.; Terwilliger, T. C.; Smith, R. D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7022-7027. (27) Greig, M. J.; Gaus, H.; Cummins, L. L.; Sasmor, H.; Griffey, R. H. J. Am. Chem. Soc. 1995, 117, 10765-10766.

MATERIALS AND METHODS Peptides and RNA. TAR RNA samples were chemically synthesized using phosphoramidite chemistry, purified by polyacrylamide gel electrophoresis, and characterized by enzymatic sequencing and ESI mass spectrometry.28 Gel-purified RNA samples for ESI-MS binding studies were further desalted by cold ethanol precipitation as ammonium acetate salts.29,30 RNA samples were quantitated by UV spectroscopy by measuring the absorbance at 260 nm. Recombinant Tat protein was expressed from Escherichia coli, purified by HPLC, and characterized by N-terminal Edman sequencing and ESI-MS.28 Tat peptides were synthesized by solid phase synthesis using standard Boc chemistry protocols and purified by reversed-phase HPLC. Arginine peptide, Arg11, was synthesized by the University of Michigan Protein and Carbohydrate Structure Facility (Ann Arbor, MI). Peptide concentrations were based on amino acid analysis. Protein and RNA solutions were buffered with 10-25 mM ammonium acetate, pH 6.9. Peptide solutions were buffered with 10 mM ammonium acetate, pH 6.9, and 0.01% Nonidet-P40. Nonidet-P40, a nonionic surfactant, was used to prevent the aggregation of peptides. RNA solutions were annealed by heating for 4 min at 95 °C and cooled slowly (3-4 h) to room temperature to assure that the RNA had the proper tertiary structure. For the protein-RNA experiments, methanol (10% v/v) and 1,2diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA) were added to the aqueous ammonium acetate ESI-MS solution. The addition of methanol enhanced the stability of ESI-MS signal without altering the resulting spectra to a significant extent for these systems. The adduction of cations from ubiquitous salts by RNA was reduced by ethanol precipitation of RNA and by the addition of CDTA, as prescribed by McCloskey and co-workers.30 Mass Spectrometry. ESI mass spectrometry was performed with a double-focusing hybrid mass spectrometer (EBqQ geometry, Finnigan MAT 900Q, Bremen, Germany) with a mass-tocharge (m/z) range of 10 000 at 5 kV full acceleration potential.31 A position-and-time-resolved ion-counting (PATRIC) scanning focal plane detector with an 8% m/z range of the m/z centered on the array detector was used.32 For most of the experiments, an ESI interface based on a heated glass capillary inlet was used.33 Warm nitrogen gas (∼60 °C) countercurrent to the electrospray aided droplet and ion desolvation.33 The nitrogen gas flow rate (3 L min-1) can influence the amount of residual solvation observed for the multiply charged ions as well as the sensitivity of ESI, especially for ESI from aqueous solutions. Gas phase collisions, controlled by adjustment of the voltage difference between the tube lens at the metallized exit of the glass capillary and the first skimmer element (∆VTS), were also used to augment the desolvation of the ESI-produced droplets and ions. A stream of SF6 coaxial to the spray suppressed corona discharges, especially (28) Mei, H.-Y.; Galan, A. A.; Halim, N. S.; Mack, D. P.; Moreland, D. W.; Sanders, K. B.; Truong, H. N.; Czarnik, A. W. Bioorg. Med. Chem. Lett. 1995, 5, 2755-2760. (29) Stults, J. T.; Marsters, J. C. Rapid Commun. Mass Spectrom. 1991, 5, 359363. (30) Limbach, P. A.; Crain, P. F.; McCloskey, J. A. J. Am. Soc. Mass Spectrom. 1995, 6, 27-39. (31) Loo, J. A.; Ogorzalek Loo, R. R.; Andrews, P. C. Org. Mass Spectrom. 1993, 28, 1640-1649. (32) Loo, J. A.; Pesch, R. Anal. Chem. 1994, 66, 3659-3663. (33) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71.

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Figure 1. Sequences and proposed secondary structure of (A) HIV-1 TAR31 RNA, (B) TAR28 RNA, (C) bulge linker TAR RNA, and (D) loop linker TAR RNA. (E) Sequences of HIV-1 Tat protein (residues 1-86) and peptide (Tat40, residues 45-86) used for this study.

important for ESI of aqueous solutions. Solution flow rates delivered to the ESI source were typically in the 0.5-1.0 µL min-1 range. Additionally, preliminary data were acquired on a recently installed ESI source with low analyte solution flow capabilities (150 nL min-1).34 The ESI interface is based on a heated metal capillary inlet.35 With this interface, countercurrent N2 gas is not necessary for efficient droplet and ion desolvation. The temperature of the metal capillary was adjusted to 150 °C. RESULTS AND DISCUSSION Tat Peptide Binding to TAR RNA. The amino acid sequence of Tat protein contains a basic amino acid-rich region, RKKRRQRRR (residues 49-57) that is essential for TAR RNA binding and recognition (Figure 1). Fragments of Tat protein containing this basic region, such as the C-terminal 40-residue peptide (Tat40) used in this study, specifically bind to TAR RNA with affinity similar to that of full-length Tat.28,36-38 TAR RNA is a small RNA hairpin consisting of a stem-loop structure with a pyrimidine bulge (Figure 1). The three-nucleotide bulge has been shown to be essential for Tat recognition and activity.3,39,40 An arginine at residue 52 or 53 of Tat protein is important for TAR RNA binding affinity and specificity. The other residues in the basic region can be replaced with lysine without loss of binding affinity or specificity.41 Arginine has two amino groups and a secondary amine which can form an extensive network of hydrogen bonds (34) Muenster, H.; Pesch, R.; Dobberstein, P. In Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 1216, 1996; American Society for Mass Spectrometry: Santa Fe, NM, 1996; p 1026. (35) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4, 81-87. (36) Weeks, K. M.; Ampe, C.; Schultz, S. C.; Steitz, T. A.; Crothers, D. M. Science 1990, 249, 1281-1285. (37) Frankel, A. D. Protein Sci. 1992, 1, 1539-1542. (38) Long, K. S.; Crothers, D. M. Biochemistry 1995, 34, 8885-8895. (39) Roy, S.; Delling, U.; Chen, C.-H.; Rosen, C. A.; Sonenberg, N. Genes Develop. 1990, 4, 1365-1373. (40) Cordingley, M. G.; LaFemina, R. L.; Callahan, P. L.; Condra, J. H.; Gotlib, L.; Schlabach, A. J.; Colonno, R. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 8985-8989. (41) Calnan, B. J.; Tidor, B.; Biancalana, S.; Hudson, D.; Frankel, A. D. Science 1991, 252, 1167-1171.

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Figure 2. Negative ion ESI mass spectra of the 1:1 Tat40 peptideTAR RNA complex (9, Mr ) 14 585) using (A) an ESI source with a heated glass capillary (1.0 µL min-1 analyte flow) and (B) a microESI source with a heated metal capillary (0.15 µL min-1 analyte flow). Tat40 peptide (Mr ) 4644) was added at a 1.5 molar ratio to a solution containing previously annealed TAR31 RNA (Mr ) 9941, 13 pmol µL-1), 0.3 mM CDTA, 10% (v/v) methanol, and 10 mM ammonium acetate, pH 6.9. There is less charging of the complex in the ESI source with a heated metal capillary.

with the phosphate backbone and bases of TAR RNA.41 In contrast, lysine has only one amino group with which it can form hydrogen bonds and, thus, only binds nonspecifically to TAR RNA.41 Upon binding arginine peptides, a local conformational rearrangement in the bulge region of TAR RNA occurs.6 A specific spatial arrangement of phosphate groups (P21, P22, and P40) and base functional groups (G26-N7, A27-N7, U23-O4, and N3) of TAR RNA is created that can interact with amino acid side chains from Tat protein and increase binding affinity and specificity; i.e., free- and bound-TAR have distinct conformations.6,42 Therefore, the tertiary structure of TAR RNA is important for Tat recognition. Negative ion ESI-MS of a solution containing 40 amino acid Tat peptide (Tat40, Mr ) 4644) and TAR RNA (TAR31, 31-mer, Mr ) 9941) yields a 14.5 kDa peptide-RNA complex, consistent with a 1:1 stoichiometry (Figure 2A). Gel mobility shift assays have shown that the Tat40 peptide binds specifically to the TAR 31-mer RNA with an apparent dissociation constant (Kd) of ∼1 nM.28 This value is consistent with dissociation constants measured for other Tat-derived peptides.36 At very high concentrations of Tat40 peptide, 2:1 and 3:1 peptide-RNA complexes are observed as the maximum stoichiometry (Figure 3). Multiple binding of Tat to TAR has been observed previously using other biophysical techniques. Using NMR, Aboul-ela and co-workers showed that there is some evidence for binding of a second Tat peptide (residues 37-72, 32 residues) to the lower stem of TAR 29-mer (residues 17-45).6 Gel mobility assays show that multiple binding of a Tat peptide (38 residues) to the full-length TAR RNA (57 residues) occurs but not to a shorter TAR RNA (27 residues).36 It is likely that the initial peptide-RNA binding event is specific in nature, and the following interactions are classified as nonspecific events that occur in solution and possibly in the gas phase. (42) Aboul-ela, F.; Karn, J.; Varani, G. Nucleic Acids Res. 1996, 24, 3974-3981. (43) Sannes-Lowery, K. A.; Mack, D. P.; Hu, P.; Mei, H.-Y.; Loo, J. A. J. Am. Soc. Mass Spectrom. 1997, 8, 90-95.

Figure 4. Positive ion ESI mass spectrum of the 1:1 Tat protein (86 residues, Mr ) 9778)-TAR31 RNA complex in 10 mM ammonium acetate, pH 6.9, 10% (v/v) methanol, and 1 mM DTT. 9, Tat-TAR complex and b, free TAR RNA.

Figure 3. Titration graphs of TAR31 RNA with Tat40 peptide using (top) positive ion ESI-MS and (bottom) negative ion ESI-MS. b, TAR RNA; 9, 1:1 Tat-TAR complex; [, 2:1 Tat-TAR complex; and 2, 3:1 Tat-TAR complex.

Previously, we have shown that positive ion ESI-MS of DNA and RNA molecules is possible.43 In solution, all oligonucleotides have a net negative charge and can form noncovalent complexes with positive counterions such as ammonium ions. Desorption of these noncovalent complexes into the gas phase, followed by collisionally activated dissociation (CAD), leads to positively charged RNA molecules by neutralization of the phosphate groups on the sugar backbone and protonation of some of the bases. Results similar to the negative ion mass spectra shown in Figure 2 are obtained for positive ion ESI-MS of a Tat peptide-TAR RNA solution (Figure 3). However, at low concentrations of Tat peptide, the amount of free TAR RNA observed differs between positive and negative ion ESI-MS because RNA is more easily ionized in the negative ion mode. Positive and negative ion ESI-MS should give analogous results if the mass spectral data reflect solution phase interactions. Furthermore, after addition of acid to the Tat peptide-TAR RNA solution, dissociation of the complex was observed in both positive and negative ion ESI-MS. This is consistent with solution phase behavior because acidic conditions denature RNA and disrupt the interaction between peptides and RNA. A relatively small volume of methanol (10% v/v) was added to the RNA-peptide solution to increase stability of the electrospray and reproducibility of the data. From duplicate measurements of titration experiments such as that shown in Figure 3, the relative error limits (or level of reproducibility) is estimated to be (5%. Although organic cosolvents can affect the protein-RNA affinity for many systems, the ESI-MS data for the Tat-TAR system obtained with 10% methanol were quantitatively similar to data acquired from 100% aqueous solutions (data not shown). Studies on the Tat protein binding to TAR RNA by Fong et al.44 show that specific binding actually increases in the presence of mild (44) Fong, S. E.; Smanik, P.; Thais, T.; Smith, M. C.; Jaskunas, S. R. J. Virol. Methods 1997, 66, 91-101.

denaturing conditions (e.g., 6 M urea). In fact, abundant ESIMS signals for the Tat peptide-TAR RNA complex from a 0.5 µM concentration solution with 77% (v/v) methanol was observed (data not shown). However, further studies are needed to determine the effect of high concentrations of organic cosolvents on the specificity of the interaction. The positive ion ESI-MS of a solution containing full-length Tat protein (86 residues, 9.8 kDa) and TAR31 RNA yields a 1:1 protein-RNA complex, as shown in Figure 4. From ESI-MS of Tat protein in an acid-buffered solution, the measured protein molecular mass was determined to be 9778.3 ( 1.2 Da, whereas the molecular mass calculated from its sequence is 9784.3 Da. The measured molecular mass of Tat protein after addition of the reducing agent tris(2-carboxyethyl)phosphine (TCEP) was 9784.0 ( 1.1 Da.28 The seven cysteine residues can form three intramolecular disulfide bonds, accounting for the 6 Da mass difference between the oxidized and reduced forms of the protein. The addition of a reducing agent (e.g., dithiothreitol, DTT) to the pH 6.9 solution was necessary for observing the Tat protein-RNA complex. Apparently, Tat protein needs to be in the disulfidereduced state to form a complex with TAR RNA. After addition of acid to the solution, only the Tat protein was observed, as expected. Weakly bound complexes can be dissociated in the ESI atmospheric pressure/vacuum interface by adjustment of ∆VTS. No experimental conditions were found to dissociate the gas phase 1:1 peptide-RNA complex, even for peptide-RNA complexes with lower binding affinities. Protein-oligonucleotide interactions composed mostly of electrostatic forces are likely to be extremely stable in a solvent-less environment. Electrostatic interactions may be strengthened in vacuum, as Coulombic stabilization of opposite charges may be enhanced. Feng studied the binding of highly basic spermine to an acidic spermine-binding peptide (SBP, 1607 Da) containing four glutamic acid residues.45 Despite a weak binding constant (104 M-1) in solution, the spermine-SBP complex could be readily detected by ESI-MS. Moreover, the complex is unusually stable to gas phase CAD. Studies of (45) Feng, R. In Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, May 21-26, 1995; American Society for Mass Spectrometry: Santa Fe, NM, 1995; p 1264.

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protein-DNA complexes25,26 and the Tat-TAR complexes shown in this report are additional examples where dissociation of the gas phase complex is very difficult; i.e., the multiply charged ions for the complex are stable at high interface energies. Dissociation of covalent bonds from either the RNA or peptide is observed at high collision energies, rather than dissociation of the complex. Preliminary ESI-MS experiments with the Tat-TAR peptideRNA complex were performed with a recently installed heated metal capillary ESI interface fitted with a low-flow micro-ESI source. The initial results (Figure 2B) show good signal-to-noise ratio for ions of the 1:1 Tat40-TAR31 complex (with nearly onetenth the sample consumed), although the observed charge distribution appears to be shifted to lower charge relative to the glass capillary interface results (Figure 2A). More studies are necessary to determine the cause of this charge shift. Tat Peptide Binding to TAR RNA Mutants. Although the results from ESI-MS experiments are believed to reflect solution phase interactions, this cannot be taken for granted. Control experiments must be done to assure that the complexes observed in the mass spectra represent specific interactions as opposed to gas phase aggregation.18,46,47 To test whether the specificity of the Tat peptide-TAR RNA interaction is mirrored by the ESIMS data, binding experiments with TAR31 mutants were performed. Because solution phase methods have demonstrated that the UCU bulge of TAR RNA is essential for Tat binding, the TAR31 mutants studied were a TAR28 RNA with the three-nucleotide UCU bulge removed, TAR with the bulge region replaced by a poly(ethylene glycol) (PEG) linker, and TAR with a PEG-substituted loop region (Figure 1). In separate binding experiments between Tat40 peptide and bulgeless TAR28 RNA, a 1:1 Tat peptide-TAR28 RNA complex as well as a small amount of free TAR28 RNA is observed (Figure 5A,B). In comparison with the binding experiments between Tat40 peptide and TAR31 RNA, it is difficult to tell whether Tat has a higher binding affinity for TAR31 RNA or for TAR28 RNA. However, relative binding affinities can be determined from competitive binding experiments using ESI-MS.25,47 Under competitive binding conditions where the total RNA concentration equals the protein concentration in solution, ESI mass spectra show that Tat40 peptide affinity is greatly reduced for the bulgeless TAR28 RNA (Figure 5C), consistent with solution phase measurements.39 As indicated in the mass spectrum, the amount of free TAR28 is greater than that of free TAR31, and the relative abundance of bound TAR31 is greater than the abundance of bound TAR28. Reduced affinity of Tat40 peptide to PEG-bulge TAR RNA relative to TAR31 RNA also was observed by competitive binding ESI-MS experiments, consistent with gel mobility shift experiments.28 On the other hand, competitive binding experiments showed that Tat peptide has nearly equal affinity for the PEGloop TAR RNA and the TAR31 RNA, consistent with previously reported structural data;28 the loop region is not intimately involved with peptide binding and is believed to be involved with other protein factors. Thus, ESI-MS data confirm that the bulge region of TAR RNA is critical to the binding affinity of Tat peptide. (46) Smith, R. D.; Light-Wahl, K. J. Biol. Mass Spectrom. 1993, 22, 493-501. (47) Loo, J. A.; Hu, P.; McConnell, P.; Mueller, W. T.; Sawyer, T. K.; Thanabal, V. J. Am. Soc. Mass Spectrom. 1997, 8, 234-243.

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Figure 5. (A) Deconvoluted negative ion ESI mass spectrum from a solution containing 2 molar equivalents of Tat40 peptide to 1 molar equivalent of TAR31 RNA (10 µM) in 10 mM ammonium acetate, pH 6.9. There is no free TAR31 RNA. (B) Deconvoluted negative ion ESI mass spectrum from a solution containing 2 molar equivalents of Tat40 peptide to 1 molar equivalent of TAR28 RNA (no bulge, 10 µM) in 10 mM ammonium acetate, pH 6.9. The inset shows the amount of free TAR28 RNA. (C) Deconvoluted negative ion ESI mass spectrum from a solution containing 2 molar equivalents of Tat40 peptide to 1 molar equivalent of TAR31 RNA (10 µM) to 1 molar equivalent of TAR28 RNA (no bulge) in 10 mM ammonium acetate, pH 6.9. Tat40 peptide preferentially binds to TAR31 RNA under competitive binding conditions.

Titration experiments with ESI-MS detection have been used to measure absolute binding affinities.27,47,48 However, all of these reported studies involved macromolecular complexes with micromolar affinities. The Tat-TAR system is currently not amenable to such studies because of its nanomolar binding constants; the range of ligand concentrations should be above and below the dissociation constant. It is hoped that improvements in ESIMS sensitivity (e.g., low-flow nanoelectrospray49) will allow for the analysis of much lower concentration solutions. Regardless of these limitations, it is demonstrated that relative binding affinities can be determined from competition experiments. Tat-TAR Complex Dissociation by Arg-Containing Peptide. Because the complexation of Tat protein to TAR RNA is critical for replication of HIV, compounds that interfere with this complexation may exhibit antiviral activity.50 For example, Hamy et al. reported on a nine-residue peptoid that specifically inhibits Tat-TAR interactions.51 ESI-MS provides an ideal method to determine drug binding stoichiometry and, potentially, binding constants for biochemical systems. Calnan et al.52 showed that Arg9 binds TAR RNA with the same affinity and specificity as the Tat peptides. Thus, the peptide Arg11 should also effectively compete with the Tat peptide for binding TAR RNA. Figure 6 (48) Lim, H.-K.; Hsieh, Y. L.; Ganem, B.; Henion, J. J. Mass Spectrom. 1995, 30, 708-714. (49) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (50) Mei, H.-Y.; Mack, D. P.; Galan, A. A.; Halim, N. S.; Heldsinger, A.; Loo, J. A.; Moreland, D. W.; Sannes-Lowery, K. A.; Sharmeen, L.; Truong, H. N.; Czarnik, A. W. Bioorg. Med. Chem. 1997, 5, 1173-1184. (51) Hamy, F.; Felder, E. R.; Heizmann, G.; Lazdins, J.; Aboul-Ela, F.; Varani, G.; Karn, J.; Klimkait, T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 35483553. (52) Calnan, B. J.; Biancalana, S.; Hudson, D.; Frankel, A. D. Genes Develop. 1991, 5, 201-210.

Figure 6. (A) Positive ion ESI mass spectrum of the 1:1 Tat40 peptide-TAR31 RNA complex. Tat40 peptide was added at a 1.5 molar ratio to a solution containing previously annealed TAR31 RNA (13 pmol µL-1), 0.3 mM CDTA, 10% (v/v) methanol, and 10 mM ammonium acetate, pH 6.9. (B) Positive ion ESI mass spectrum from a solution containing Tat40 peptide, TAR RNA (13 pmol µL-1), and Arg11 (Mr ) 1736) at a molar ratio of 1.5:1:2, respectively, in 0.3 mM CDTA, 10% (v/v) methanol, and 10 mM ammonium acetate, pH 6.9. Addition of Arg11 results in dissociation of the Tat-TAR complex. b, TAR RNA; 9, 1:1 Tat-TAR complex; [, 2:1 Tat-TAR complex; O, 1:1 TAR-Arg11 complex; and 2, 1:1:1 Tat-TAR-Arg11 complex.

shows the positive ion ESI mass spectrum that resulted from the addition of Arg11 to a solution containing Tat40 peptide and TAR31 RNA. Dissociation of the Tat-TAR complex is evident by the ions representing the Arg11-TAR complex. In addition, the ternary Tat-TAR-Arg11 complex was also observed. Titration of the 1:1 Tat-TAR complex with Arg11 shows the ternary TatTAR-Arg11 complex dominating at high concentrations of Arg11 (Figure 7). As discussed for the higher order Tat peptide-TAR RNA complex, it is likely that the lower stem region of TAR provides an additional binding site to accommodate a ternary complex.6 Negative ion ESI-MS confirms the dissociation of the Tat-TAR complex by formation of the Arg11-TAR complex and confirms the behavior of Arg11 as an effective competitive inhibitor against Tat40 peptide. CONCLUSIONS Many examples in the literature have demonstrated that ESIMS can be used to measure specific noncovalent interactions.18 In particular, reports of ESI-MS gas phase binding constant measurements for selected systems, which were in reasonable

Figure 7. Titration graph of the 1:1 Tat40-TAR31 complex with Arg11, with initial peptide-RNA concentration of 13 pmol µL-1. The ternary Tat-TAR-Arg11 complex dominates. 9, 1:1 Tat-TAR complex; [, 2:1 Tat-TAR complex; O, 1:1 TAR-Arg11 complex; and 2, 1:1:1 TatTAR-Arg11 complex.

agreement with solution phase values, have appeared.27,47,48 Our studies indicate that specific interactions between peptides and RNAs can be maintained in the gas phase and observed by mass spectrometry. The potential use of mass spectrometry for studying the behavior of compounds that target specific protein-RNA recognition events is promising. However, it is still not clear what types of molecular interactions can be studied with present day MS methods.18 Do factors such as solution phase dissociation constants or the bonding type (e.g., electrostatic, hydrophobic, etc.) that govern noncovalent interactions affect the “success” of the ESI experiment? What experimental precautions are necessary to ensure good fidelity between the gas phase measurement and the solution phase behavior? As the methodology matures, more research laboratories will be using electrospray ionization MS to study important weakly bound complexes. ACKNOWLEDGMENT We acknowledge Margaret Whitton and Scott Buckel for N-terminal sequencing of the Tat protein.

Received for review July 11, 1997. Accepted September 30, 1997.X AC970745W X

Abstract published in Advance ACS Abstracts, November 15, 1997.

Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

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