J. Am. Chem. SOC.1993,115, 63336345
6333
Characterization of Amino Acid Side Chain Dynamics in a Zinc-Finger Peptide Using 13CNMR Spectroscopy and Time-Resolved Fluorescence Spectroscopy Arthur G. Palmer, III,? Remo A. Hochstrasser, David P. Millar, Mark Rance, and Peter E. Wright' Contributionfrom the Department of Molecular Biology, The Scripps Research Institute. La Jolla, California 92037 Received September 23, 1992
Abstract: Proton-detected 13C NMR spectroscopy has been used to measure spin-lattice and spin-spin relaxation rate constants and the steady-state (1Hj-13C nuclear Overhauser effect enhancements for the protonated aromatic carbon spins and for the methyl carbon spins in the zinc complex of a single zinc-finger peptide. Measurements were performed at 11.7 T for aromatic spins and at 7.0 and 11.7 T for methyl spins. Time-resolved fluorescence spectroscopy has been used to measure the polarization anisotropy decay for the single tyrosyl fluorophore in the peptide in the presence and absence of zinc. The NMR relaxation and fluorescencedepolarization data are analyzed using a model-free formalism in which internal motions are characterized by order parameters and effective correlation times. Order parameters for Tyr and His aromatic moieties are similar to the order parameters for the corresponding backbone a carbon spins reported previously (Palmer, A. G.; Rance, M.; Wright, P. E. J. Am. Chem. SOC.1991, 113, 4371-4380); thus, these aromatic side chains have little additional conformational freedom on picosecond to nanosecond time scales. In contrast, order parameters for Phe 10 indicate that the phenyl ring is more highly restricted than the backbone a carbon spin; thus, the order parameters reflect the packing of the Phe 10 side chain in the hydrophobic core of the molecule. Phenomenological spin-spin relaxation rate constants for the two His residues provide evidence that a chemicalexchange process occurs in the zinc-binding site of the peptide. Order parameters for the symmetry axes of the methyl groups are determined from the methyl C-H order parameters assuming tetrahedral geometry. In general, order parameters for the symmetry axes are smaller for residues with longer side chains; however, the order parameters for Leu 16 have nearly maximal values and reflect the packing of Leu 16 in the hydrophobic core of the peptide. The order parameters for the symmetry axes of the geminal methyl groups of Val and Leu residues are compared to order parameters obtained previously for the Val j3 and Leu y methine carbon spins; the observed differences may reflect steric constraints on motions of the methyl and methine groups. The symmetry axis order parameter for Ala 15 is larger than unity and indicates that relaxation is mediated by motions with effective correlation times in excess of 100 ps. Analysis using specific models for internal motions of methyl groups suggests that the motion of this methyl group cannot be described simply by restricted rotational diffusion or three-site jump models. Order parameters measured by NMR and fluorescence spectroscopies for the tyrosyl ring in the zinc complex of the peptide are in good agreement; consequently, order parameters appear to be accurately determined by both experimental techniques. The overall rotational correlation time measured by fluorescence spectroscopy is 19% smaller than that measured by NMR spectroscopy. Fluorescencedepolarization is faster in the absence of zinc and probably is dominated by large amplitude local motions of the unfolded peptide.
Introduction Nuclear magnetic spin relaxation and fluorescence depolarization are coupled to stochastic overall and internal motions of molecules in solution. Consequently, nuclear magnetic resonance (NMR)lJ and fluorescence spectroscopies3~4are powerful techniques for experimental investigation of dynamics at individual sites in molecules. Characterization of motional phenomena in biological macromolecules is critical for a detailed biophysical understanding of function and complements determination of the time-averaged three-dimensional structures by X-ray crystallography and NMR spectroscopy. Direct measurementsof 13Cand 15Nspin relaxationin biological macromolecules are hindered by the small gyromagnetic ratios of these nuclei and by the limited resolution of one-dimensional NMR techniques. Proton-detected two-dimensional heteronuclear NMR spectroscopy provides significant improvements in Presentaddress: Department of Biochemistry and MolecularBiophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032. (1) London, R. E. Methods Enzymol. 1989,176, 358-375. (2) Williams, R. J. P. Eur. J . Biochem. 1989,183,479497. ( 3 ) Beechem, J. M.; Brand, L. Annu. Rev. Biochem. 1985, 54,43-71. (4) Bucci, E.; Steiner, R. F. Biophys. Chem. 1988, 30, 199-224.
sensitivity and resolution and has rejuvenated the study of 13C and I5Nspin relaxation in biological macromolecules.5-8 Characterizationof 13Crelaxationis particularly useful for investigation of dynamics in peptides and proteins because carbon atoms are distributed along the amino acid backbones and throughout side chains;g-12 nonetheless, investigations of backbone dynamics of proteins using l5N NMR spectroscopy have been more common because of the availability of isotopically enriched ~pecies.~3-20 (5) Kay, L. E.; Jue, T. L.; Bangerter, B.; Demou, P. C. J. Mag.Reson. 1987, 73, 558-564. (6) Sklenar, V.; Torchia, D.; Bax, A. J. Magn.Reson. 1987,73,375-379. (7) Nirmala,N. R.; Wagner,G. J.Am. Chem.Soc. 1988,110,7557-7558. (8) Nirmala, N. R.; Wagner, G. J. Magn.Reson. 1989,82, 659-661. (9) Dellwo, M. J.; Wand, A. J. J. Am. Chem. Soc. 1989.11 1,45714578. (10) Palmer, A. G.; Rance, M.; Wright, P. E. J. Am. Chem. Soc. 1991, 113,4371-4380. (1 1) Kelsh, L. P.; Ellena, J. F.; Cafio, D. S. Biochemistry 1992,31,51365144. (1 2) Nicholson, L. K.; Kay, L. E.; Baldisseri, D. M.; Arango, J.; Young, P. E.; Torchia, D. A. Biochemistry 1992, 31, 5253-5263. (13) Kay, L. E.; Torchia, D. A.; Bax, A. Biochemistry 1989, 28, 89728979. (14) Clore, G. M.; Driscoll, P. C.; Wingfield, P. T.; Gronenborn, A. M. Biochemistry 1990,29, 7387-7401. (15) Barbato,G.;Ikura, M.;Kay, L. E.;Pastor,R. W.;Bax, A. Biochemistry 1992,31, 5269-5278.
0002-7863/93/1515-6333$04.00/00 1993 American Chemical Society
6334 J. Am. Chem. Soc., Vol. 115, No. 14, 1993 Time-resolved decay of the polarization anisotropy of fluorescence emission has been used extensively to examine motional propertiesof intrinsicand extrinsicfluorescentprobes in proteins.314 Fluorescence spectroscopy is more sensitive than NMR spectroscopy and directly monitors dynamic processes on picosecond and nanosecond time scales; however, the number of molecular sites available for investigation is more limited. Relatively few investigations of intrinsic fluorescence in proteins have utilized the Tyr fluorophore,21-23 because Tyr fluorescence usually is quenched by nearby Trp residues. In addition, few investigations have been reported that compare motional parameters obtained from NMR and fluorescence spectroscopies.24J5 The intramolecular dynamics of the zinc complexof a 25 amino acid zinc-finger peptide, Xfin-3 1, have been characterized previously for the backbone Ca spins and for the side chain Val C@and Leu Cy spins.1° In the present study, recently developed techniques for proton-detected 13Crelaxation measurements are used to quantify relaxation parameters for the protonated carbons of the Tyr, Phe, and His aromatic moieties and the terminal methyl carbon spins of the Ala, Val, and Leu residues. Timeresolved fluorescence spectroscopy is used to measure the polarization anisotropy decay of the single Tyr fluorophore in Xfin-31 in the presence and absence of zinc. NMR and fluorescenceresults are interpreted using both model-freemethods and specificmotionalmodels for internal dynamics. A comparison of the experimental NMR results with the results of molecular dynamics simulations is reported elsewhere.26
Theory NMR spin relaxation parameters are measured using twodimensionalproton-detected analogs of the inver~ion-recovery,~~ Carr-Purcell-Meiboom-Gill (CPMG),2829and steady-state nuclear Overhauser effect (NOE) experiments.30 Ignoring crosscorrelation between dipolar and chemical shift anisotropy (CSA) relaxation mechanisms31 and cross-correlation between methyl bond vectors,92 the spin-lattice relaxation rate constant, spinspin relaxation rate constant, and steady-state (lHj_13C NOE enhancement are given by, r e ~ p e c t i v e l y , ~ ~ . ~ ~
Palmer et al. of interest; TH and y c are the gyromagnetic ratios of the proton and 13C spins, respectively; rCH is the length of the C-H bond vector; OH and oc are the Larmor frequencies of the lH and l3C spins, respectively; Au is the chemical shift anisotropy; h is Planck's constant divided by 2u;and w is the permittivity of free space. For an axially symmetricchemical shift tensor with parallel and perpendicular components denoted ull and uL, respectively, At7 = ul - uL. In eq 2, R, is the systematic contribution to Rz from processes, such as chemical exchange, that lead to dephasing of the 13C magnetization during the CPMG pulse train; R. is significant only if ks > 1, in which k is the rate constant of the dephasing mechanism and 2s is the pulse spacing in the CPMG sequence.35 The auto-spectral density function, J ( w ) , is the Fourier transform of the orientational autocorrelation function, C(s),
J ( w ) = 2JOmC(r)COS
07
ds
(6)
with
c(7)= (y:[n(o)ly:[~(~)1)
= I/5(P2[4(O)*Pr(7)I) (7)
in which Yzo[Q(t)] is a modified spherical harmonic function,36 P2(x) is the second Legendre polynomial, n(t) are the polar angles specifying the orientation in the laboratory frame of a unit vector, ~ ( t )that , is defined by the relevant relaxation mechanism, and ( ) indicates an ensemble average. For dipolar relaxation, ~ ( t ) points along the C-H bond; for CSA relaxation by a symmetric chemical shift tensor, ~ ( t points ) along the principal axis of the tensor.34 In a time-resolved fluorescence depolarization experiment, fluorophores in the sample are excited by a laser pulse that propagates along the y-axis and is polarized in the z-direction, in the laboratory reference frame. The intensities of the componentsof the emitted fluorescencethat are polarized parallel, Zll(t), and perpendicular, ZL(t), to the z-axis are measured in the x-direction as functions of the time following the pulse, t . The fluorescence polarization anisotropy is given by r(t) = [Z#) - ZJ~)I/[Zl@
+ 2Z,(t)l
(8)
If unit vectors along the directions of the absorption and emission dipole transition moments, p.(t) and ae(t), respectively, are parallel, or if the fluorophore is axially symmetric about be(t), then
npis the number of protons directly attached to the carbon atom (16) KBrdel, J.; Skelton, N. J.; Akke, M.; Palmer, A. G.; Chazin, W. J. Biochemistry 1992, 31, 4856-4866. (17) Peng, J. W.; Wagner, 0. Biochemistry 1992, 31, 8571-8586. (18) Schneider, D. M.; Dellwo, M. J.; Wand, A. J. Biochemistry 1992,31,
3645-3652. .. . . (19) Stone, M. J.; Fairbrother, W. J.; Palmer, A. G.; Reizer, J.; Saier, M. H.; Wright, P. E. Biochemistry 1992, 31, 43944406. (20) Stone, M. J.; Chandrasekhar,K.;Holmgren,A,;Wright, P. E.; Dyson, H. J. Biochemistry 1993, 32, 426-435. (21) Hgrd,T.;Hsu,V.;Sayre,M. H.;Geiduschek,E.P.;Appclt,K.;Keams, D. R. Biochemistry 1989,28, 396-406. (22) Lakowicz, J. R.; Laczko, G.; Gryczynski, I. Biochemistry 1987, 26, 82-90. (23) Rigler, R.; Roslund, J.; Forsen, S. Eur. J. Biochem. 1990,188, 541545. (24) Weaver, A. J.; Kemple, M. D.; Prendergast, F. G. Biophys. J . 1988,
-$4., 1-15 . .-.
(25) Weaver, A. J.; Kemple, M. D.;Prendergast, F. G. Biochemistry 1989, 28.86144623. (26) Palmer, A. G.; Case, D. A. J . Am. Chem.SOC.1992,114,9059-9067.
in which X is the constant angle between ~ ( t and ) &), and ro is the limiting polarization ani~otropy.3~As shown by eq 9, ro I 0.4, and the equality is satisfied if fi, and ccc are parallel. The formal correspondencebetween NMR spin relaxation and fluorescence depolarization is indicated by the right-hand sides of eqs 7 and 9: both phenomena depend upon molecular motions through an autocorrelation function of the form (P2[fi(O)u(s)] ). In the following, the motions of a unit vector b(r) attached to an (27) Vold, R. L.; Waugh, J. S.; Klein, M. P.; Phelps, D. E. J. Chem. Phys. 1968,48, 3831-3832. (28) Cam, H. Y.; Purcell, E. M. Phys. Rev. 1954, 94, 630-638. (29) Meiboom, S.; Gill, D. Rev. Sci. Instrum. 1958,29,688-691. (30) Noggle, J. H.; Shirmer, R. E. The Nuclear Overhauser Effect: Chemical Applications; Academic Press: New Yorlt, 1971. (31) Goldman, M. J. Magn. Reson. 1984, 60, 437-452. (32) Werbelow, L. G.;Grant, D. M. Adv. Magn. Reson. 1977,9,189-299. (33) Abragam, A. Principles of Nuclear Magnetism; Clarendon Press: Oxford, U.K. 1961. (34) Lipari, G.; Szabo,A. J. Am. Chem. Soc. 1982,104,4546-4559, (35) Bloom, M.; Reeves, L. W.; Wells, E. J. J. Chem. Phys. 1965, 42, 1615-1624. (36) Brink, D. M.; Satchier, G. R. Angular Momentum; Clarendon Press: Oxford, U.K. 1968. (37) Lipari, G.; Szabo,A. Biophys. J. 19%0, 30, 489-506.
Amino Acid Side Chain Dynamics in a Zinc-Finger Peptide amino acid side chain are described by time-dependentcoordinate transformations first from the laboratory frame to a fixed molecular reference frame M and then from frame M to an internal frame D in which the orientation of the vector is constant. Assumingthat overallrotational motion of the molecule is isotropic and independent of internal motions, the autocorrelation function is given by
= (Pz[IL(O).M(T)I
) 2
2
2
x
= eXp[-T/T,] am-2
1-2 b -2
(Da,bZ*[S2MD(o)IDa,Y2[S2MD(T)l )
yzb*
[@DFdDF]y:[@DF,4DFl
6b-b’,kJV
(lo)
in which T~ is the overall rotational correlation time constant for the molecule, Da,b2[nMD(r)] are Wigner rotation matrices,36 the time-dependent Euler angles QMD(f) = {(TMD(f),gMD(f),YMD(?)} describe the motions of the D frame relative to the M frame, Y2b[@DF,f$DF] are modified spherical harmonic functions,36 and { ~ D F , ~ D Fare ) the constant polar angles defining the orientation of I(?) in the D reference frame. The Kronecker delta function, 6w,tN, with k and N integers, expresses the requirement that G(T)be invariant under rotation by 27r/N if the molecular group that contains p ( t ) has N-fold rotational symmetry about the z-axis of the D frame. Evaluation of eq 10 for a time satisfying the inequalities T~ Cc = C6. The high order parameters observed for the aromatic spins of Phe 10 and for the symmetry axes of the methyl groups of Leu 16 (see below) are consistent with the closecontacts observed between these twosidechains in theNMR structure of the peptide and support the conclusion that these side chains form a small but stable hydrophobiccore.62 However, since only single resonances are observed for the 6 and Q spins of Phe 10 (Figure 2a), movements of the phenyl ring or the Leu side chain must occur to permit flipping of the aromatic ring on a time scale that is slow compared with rotational motion but fast compared with the chemical shift differences between the symmetry-related spins. The very large values of R,obtained for the His residues supports the previous conclusionthat a chemicalexchange process occurs in the zinc-binding site. Interestingly, only one of the two protonated 13Cspins for each His is affected by exchange broadening, and the affected spin is different for the two His residues; consequently, the chemical shift difference between conformers must be large only for one of the protonated carbon nuclei on each ring. Since R, is only appreciable if kr > 1, the rate constant for chemical exchange is >2000 s-l. Because chemical shift tensors have not been measured experimentallyfor the protonated aromatic carbon spins in amino acids, the values of Au (Table I) used in eq 5 were estimated from results for other aromatic molecules and from theoretical c a l ~ u l a t i o n s . 6 The ~ ~ values of u22 and 6 3 3 in Table I are not equal, and the theoretical descriptions appropriate for an axially symmetric shift tensor are not strictly applicable. In addition, the principal axis of the chemical shift tensor for 13C spins in aromatic moieties is oriented approximately perpendicular to the plane of the aromatic ringas (@DF = 90° for Tyr and Phe); thus, the autocorrelation functions for the dipolar and CSA relaxation mechanisms are not necessarily equal (eq 13). However, since G/DZ = 0.2 (eqs 4 and 5), relaxation is dominated by the dipolar interaction, and complications arising from the description of the CSA relaxation do not dramatically affect the given results. Altering An for an aromatic spin by -20 ppm changes the order parameter by -0.05, which is comparable to the reported uncertainties in theorder parameters (Table IV). Consequently, the conclusionsderived from comparison of the order parameters for the aromatic spins do not depend upon the exact values chosen for Au; nonetheless, more detailed interpretation of small differences in order parameters for aromatic spins awaits more (63) Moore, A. C. Ph.D. Thesis, University of California, Berkeley, CA,
1976.
(64) Prado, F. R.;Giasner-hettre, C. J. Magn. Reson. 1982, 47, 103117. (65) Veeman, W. S . Prog. Nucl. Magn.Reson. Spectrosc. 1984,16,193-
235.
J. Am. Chem. SOC.,Vol. 115, No. 14, 1993 6343 completemeasurementsof chemical shift anisotropiesfor aromatic amino acids. As a consequence of internal rotation of the methyl group, order parameters for methyl l3C spins are approximately an order of magnitude smaller than order parameters for backbone Ca spins and effective correlation times are in the range 10-25 ps, with the exception of the correlation time for Ala 15 C@(Tables V and VI). In accordance with results obtained previously for the side chain methine carbon spins (Table XI), the order parameters for the methyl groups generally are smaller for residues with longer side chains. The symmetry-axis order parameters for Leu 16 C* spins are approximately unity and, as discussed above, reflect the packing of the side chains of Phe 10 and Leu 16 in the hydrophobic core of the molecule. As shown by comparison of Tables V and VI with Table XI, Leu and Val residues can be classified according to the relationships between the symmetry-axisorder parameters for the geminal methyl spins and the order parameters for side chain methine spins. For example, all three order parameters for Leu 5 agree statistically; in contrast, for Val 11, the symmetry-axis order parameter for the yomethyl agrees well with the order parameter for the C@ spin, and the order parameter for 'yb is significantly larger. The results for Val 22 depend upon the choice of error function; however, either one (Table V) or both (Table VI) of the methyl symmetry-axis order parameters are larger than the methine C@ order parameter. Similarly, the two symmetry-axis order parameters for Leu 16 are significantly different, although the Cy methine order parameter is unknown. The symmetry-axis order parameter for Ala 15 CP is larger than the theoretical maximum value, which suggests that, for this residue at least, eq 19 is not applicable. The observed differences between the order parameters for geminal methyl carbons of Leu and Val residues or the anomalous order parameter for Ala 15 CP cannot be attributed to distortions of ideal tetrahedral geometry of the methyl group. Neutron diffraction studies of crystalline Ala and Va166967indicate that the angle between the symmetry axis and the C-H bond vectors isdecreased by 1-3O and that relativedistortionsbetweengeminal methyl groups are < l o in Val. Thus, the reported symmetry axis order parameters calculated from eq 19 may be underestimated by -20% however, relative differencesbetween order parameters for geminal methyl groups are much less affected by distortions of the methyl geometry. Incidentally, if @ = 6 8 9 is used rather than 7 0 9 in eq 19, the methyl symmetry-axis order parameter for the Leu 16 61 methyl group, as well as for Ala 15 CP, is significantly greater than unity. Alternatively, if internal motions of the methyl groups are not close to the extreme narrowing limit, then values of the order parameters are overestimated by the model-free analysis.34 In particular, the order parameter for the symmetry axis of a methyl group may be overestimatedby eq 19if steric interactions increase the barriers to internal rotation of the methyl group. In the present case, the model-free formalism is expected to be an accurate description for internal motions with wre < 0.3, which implies 7c < 75 ps.34 The effective correlation times determined from the model-free analysis were
