Assignment of Side-Chain 13C Resonances in Perdeuterated Proteins

Received January 13, 1995. For moderately sized '3C/'5N-labeled proteins, side-chain 13C resonance assignments are obtained from either the HCCH-...
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J. Am. Chem. SOC.1995,117, 4187-4188

Assignment of Side-Chain 13C Resonances in Perdeuterated Proteins


spectrum has yielded >95% of all previously unassigned sidechain 13Cresonances in 2H-HCA II. We have estimated the rotational correlation time (z,) for HCA 11 to be -10.4 ns based on the zc value for the TRP repressor-DNA complex5~*(tc 13.4 ns, 37 m a ) . The TZ relaxation times for 'HN, 15N, and non-methyl 13C spins have been calculated for 'H- and :!H-HCA II?-" The effective 13C relaxation time during the spin lock is that for magnetization, permanently resident on a non-Gly 13Ca spin, subjected to a DIPSI-3 pulse train.l2 The non-selective Tl(I3CD)relaxation time for 13Ca spins has been qualitatively measured by ID saturation recovery to be -2.6 s for :!H-HCA 11. This is significantly shorter than the Tl(l3CD)= 8.6 s calculated from intramoleculardipolar and CSA relaxation mechanisms, indicating that these mechanisms do not contribute significantly to 13C relaxation in 2H-HCA II. The nonselective Tl('Hc) is taken as -1 s; the upper limit for T2('Hc) is set to T:!('HN). These relaxation parameters allow us to estimate the theoretical gain in sensitivity, R,, for the C(CC)(CO)NH experiment applied to 2H-HCA 11 over that for the HC(CC)(CO)NH experiment applied to 'H-HCA 11. R, can be factored into a TZ contribution, R,({T:!});a T I contribution, R,({TI});and a scalar coupling contribution, Rd{Jl):


Bennett T. Farmer II**tand Ronald A. Venters*

Bristol-Myers Squibb Pharmaceutical Research Institute P.O.Box 4000, Princeton, New Jersey 08543-4000 Duke University NMR Center Duke University Medical Center P.O. Box 3711, Durham, North Carolina 27710 Received January 13, 1995 For moderately sized '3C/'5N-labeledproteins, side-chain 13C resonance assignments are obtained from either the HCCHTOCSY' or the HC(CC)(CO)NW experiment. The HCCHTOCSY has been the experiment of choice because of its extremely high sensitivity and high level of correlative redundancy. The increased number of correlation peaks generated by this experiment, however, may severely aggravate the problem of spectral overlap in larger proteins such as human carbonic anhydrase I1 (HCA 11,29 kDa, 259-residue monomer) and, in the absence of prior side-chain 13C chemical-shift assignments, may limit the utility of this experiment. Compared to 'HN resonances in /l-sheet proteins and 15N resonances in general, the dispersion of particular aliphatic 'Hc and 13C resonances is usually poorer. The HC(CC)(CO)NH experiment would therefore seem more appropriate for the assignment of side-chain 13C resonances in larger proteins. Rapid 13C and 'HN T:! relaxation, however, may render this experiment too insensitive for large proteins. In this regard, the process of sequential backbone assignments in large proteins has benefited greatly from high levels of 2H incorporation (-85%) at aliphatic site^.^-^ We have therefore adapted the HC(CC)(CO)NH experiment to perdeuterated proteins by starting the sequence of magnetization transfer steps on I3C6instead of 'Hc. In this communication, we present the results from the C(CC)(CO)NH experiment on perdeuterated HCA I1 (2H-HCA 11) and estimate the theoretical increase in sensitivity over the HC(CC)(C0)NH experiment on protonated HCA 11 ('H-HCA 11). We have previously described the preparation of HCA 11 labeled to 96% in 2Hon aliphatic sites.' Figure 1 presents the pulse sequence for the 3D gradient-enhanced and sensitivityenhanced C(CC)(CO)NH experiment. Since there are no aliphatic protons in the :!H-HCA I1 sample, the initial magnetization must originate from the side-chain 13Cspins. Figure 2 depicts 13C/15Nstrip plots corresponding to several core residues taken at the 'HN chemical shift of the respective i I residue. These data allow confirmation of both the sequential l 3 C ~ p main-chain assignments and the sequential residue type. The S / N of the peaks in Figure 2 ranges from 2:l for 1145-C,1 to 35:l for A152-Cp. The only correlation missing in Figure 2 is A152-Ca. In general, all Ala 13Cacorrelations are at best weak in the C(CC)(CO)NH spectrum. A complete analysis of this


* To whom correspondence should be addressed.

t Bristol-Myers Squibb Pharmaceutical Research Institute.

* Duke University Medical Center.

(1) Bax, A.; Clore, G. M.; Gronenbom, A. M. J. Magn. Reson. 1990,

where { } represents a set of values. R,({T1}) depends on the recycle delay TRC(1.07 s), Tl(l3CD),and Tl('Hc). It is implicit in the calculation of Rs({T2})and Rs({Jl)that the various fixed delays have been optimized for the particular experiment on HCA Il ('H vs :!H). We have calculated estimates of R,({T2}), R,({Jl), and R, for rigid-body CD/CH and CD2/CH2 aliphatic groups in 13C/ 15N-labeledHCA II. Rs({T1})evaluates to -0.5 for both groups. Rs({T:!})values are 22.4 and 26.8; R,({J)) values are 1 . 3 0 M 2.18; and R, values are therefore -3.5 and -7 for methine and methylene groups, respectively. These results indicate that the C(CC)(CO)NH experiment on 2H-HCA II is strongly favored on the basis of a large R,({T'}), which arises due to the increased T2(13CD)brought on by perdeuteration. We have also acquired an HC(CC)(CO)NH data set at 600 MHz on a 1 mM protonated 13C/15N-labeledHCA I1 sample under equally optimized experimental conditions (data not shown) for comparison with the data in Figure 2. Strip plots from the HC(CC)(CO)NH spectrum analogous to those in Figure 2 show only noise. Initial estimates of protonated side-chain 13Cchemical shifts should facilitate the analysis of the 4D HCCH-TOCSY on 'HHCA II. We have been able to reasonably predict the average one-, two-, and three-bond 2Hisotope shifts on l3Cd,4resonances in HCA 11. With a 13C resolution of f0.12 ppm in all cases, the cumulative isotope shifts, which are as large as - 1.52 ppm (Ll.QO-Cp),have been correctly estimated to within f0.25 ppm for most 13Cpresonances. Secondary structure has already been demonstrated to affect the magnitude of the 2H isotope shift on 13Ca resonance^.'^ Since secondary structure is known to alter both 13Ca and I3Cp chemical shifts from their random-coil


(2)Montelione, G.T.; Lyons, B. A.; Emerson, S. D.; Tashiro, M. J. Am. Chem. SOC. 1992,114,10974-10975. (3)LeMaster, D.M.; Richards, F. M. Biochemistry 1988,27,142-50. (4)Grzesiek, S.;Anglister, J.; Ren, H.; Bax, A. J. Am. Chem. Soc. 1993, 115,4369-4370. (5) Yamazaki, T.; Lee, W.; Revington, M.; Mattiello, D. L.; Dahlquist, F. W.; Arrowsmith, C. H.; Kay, L. E. J. Am. Chem. SOC.1994,116,64646465. (6)Fesik, S. W.; Eaton, H. L.; Olejniczak, E. T.;Zuiderweg, E. R. P.; McIntosh, L. P.; Dahlquist, F. W. J. Am. Chem. SOC.1990,112,886-888. (7)Venters, R.A.; Huang, C.-C.; Farmer; B. T.,II; Trolard, R.; Spicer, L. D.; Fierke, C. A. J. Biomol. NMR, in press.

(8)Kay, L. E. Personal communication. (9)Marhs, M. A.;Dayie, K. T.; Matsudairat, P.; Wagner, G. J. Magn. Reson., Ser. B , in press. (10)Constantine, K.L.; Friedrichs, M. S.; Goldfarb, V.; Jeffrey, P. D.; Sheriff, S.; Mueller, L. Proteins: Struct., Funct., Genet. 1993,15,290311. (11) Abragam, A. The Principles of Nuclear Magnetism; Clarendon Press: Oxford, England, 1961. (12)Shaka, A. J.; Lee, C. J.; Pines, A. J. Magn. Reson. 1988,77,274293. (13) LeMaster, D. M.; LaIuppa, J. C.; Kushlan, D. M. J. Biomol. NMR 1994,4, 863-870.

0002-786319511517-4187$09.00/00 1995 American Chemical Society

4188 J. Am. Chem. Soc., Vol. 117, No. 14, 1995

Communications io the Editor

., ..*,. . ,.. . ,, ,

ZH Wy~(WAL1Z~16x)$0.y

Figure 1. The 3D gradient-enhanced. sensitivity-enhanced C(CC)(CO)NH experiment: YO" pulses are represented by wide lines; simple 180' pulses, by black rectangles; and 90,24OY90, composite inversion pulses,l5 by striped rectangles. Gaussian- (solid-black) and G?-shaped" (crosshatched) selective inversion pulses are indicated as sinebell-shaped pulses. ' C H pulses selective far "C, spins arc labeled with C,. All other "CH pulses are nonselective. Complex data were collected in 11'' and in tZ,l8 with RDs for rpn = (x, y) and {rpa = (+x,-x); s = ( + l , - l ) on Ga) being stored separately. State~-TPPl'~ was employed on rp, and rp+ The total acquisition time was 70 h. Selected acquisition parameters are as follows: yBA''C,n,n~wd= 7.94 I;Hz with FLOPSY-8,'0 ttma"= 4.89 ms, fzmrx = 17.16 ms, ti = 69.8 ms, r, = 20.8 ms, zcEF = 3.0 ms, Acn = 13.5 ms, AnC = 13.5 ms. Ace = 4.55 ms. t n h = 5.6 ms, 6"h = 2.65 ms, 5 = 1.2 ms, GS= f32.0 Glcm, I= = 5.0 ms, Gq = 32.05 Glcm. 1- = 0.5 ms. and relaxation delay = 1.0 s. The phase cycle is rp, = 401). 4(-g); p2 = 8(x), 8(-x); rpr = 2(x). 2(-x); rp4 = x. -1; and rp, = x. 2(-x). x, -x, 2(x), 2(-x). 2(x). -x. x. 2(-x), x.

1 VI45


Figure 2. l3Clt5Nstrip plots from the C(CC)(CO)NH experiment: Results from several core residues with long side chains demonstrate the efficacy of the method for *H-HCA 11. The side-chain resonances past ' V u p are assigned by correlating the i "Cup and i I amide lSN/'HN chemical shifts obtained from the main-chain assignment data with the remainder of the "C correlations. The Al52ll59 strip plot illustrates the ability to determine amino acid types when significant overlap in amide ISN and 'HNchemical shifts exists. thereby resolving two otherwise ambiguous main-chain assignments. Thc boxed correlation peak in this plot is A152-CB.These data were acquired at 30.0 'C on a 1.6 mM ' W S N f H (96% 2H-labeled) HCA I1 sample in 100 mM phosphate buffer at pH 6.8. The amide protons were re-exchanged by unfolding the protein and then refolding it by standard methods."


values,14 the =H isotope shift on I3Ccp resonances may also be affected by secondary structure. This would tend t o decrease (14)Grzesiek. S.; Bax, A. J. Biomol. NMR 1993, 3, 185-204. (15) Levitl, M. H. Prog. NMR Spearosc. 1986. 18, 61-122. (16)Emsley. L.; Bodenhausen, G. Chem. Ph.w Lett. 1987, 165,469477 . .-.

(17)States. D. I.; Haberkom. R. A,; Ruben, D. I. J. Mngn. Reson. 1982, 48,286-292.

the predictive ability of a single, average 2H isotope shift. Sidechain ')C chemical shifts are in general less sensitive to secondary structure and may therefore exhibit a more uniform 2H isotope shift. W e have demonstrated in this communication a method to obtain complete side-chain "C assignments in 2H-HCA 11. On the basis of the number of side-chain correlation peaks and their I3Cchemical shifts, one can further restrict the possible residue types for an ('HN,I5N)pair and thereby increase the level of confidence in the sequential assignments. Prior knowledge of the side-chain I3Cchemical shifts in 2H-HCA I1 will he essential for the assignment of side-chain HN protons in 2H-HCA I1 and may also prove to be crucial t o the analvsis of the 4D HCCHTOCSY spectrum on IH-HCA I1 Acknowledgment. The Duke University NMR Center gratefully acknowledges grants from the NIH, the NSF, and the North Carolina Biotechnology Center. B.T.F. thanks L. Kay at the University of Toronto for the r, information on the TRP repressor-DNA complex and W. Metzler, K. Constantine. and L. MueUcr for their critical reading of the manuscript. Supplementary Material Available: Equation used to estimate R S ( { T , } )and additional experimental details and parameters (2 pages). This material is contained in many libraries on microfiche. immediately

follows this article in the microfilm version OF the journal, can be ordered from the ACS. and can be downloaded from the Internet; see any current masthead page for ordering information and Intemet access instructions. JA950120K (18) Palmer, A. G., 111; Cavanagh. I.; Byrd, R. A.; Rance, M. J. Magn. Reson. 1992, 96,416-424. (19)Marion. D.; Bax, A. J. M o p . Reson. 1989, 83. 205-211. (20) Mohebbi. A,; Shaka, A. I. Chem. Phys. Len. 1991. 178, 374-377. (21) Carlsson. U.;Henderson, L. E.; Lindskog, S. Biochim. Biophys. Acla 1973, 310, 376-387.