J. Am. Chem. SOC.

1993, 115, 2577-2580 2571

NMR Studies of a Cobalt-Substituted Zinc Finger Peptide?

Laura V. Harper, Barbara T. Amann, Valda Kilfoil Vinson, and Jeremy M. Berg'
Contribution from the Department of Biophysics and Biophysical Chemistry, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21 205
Received February 27, 1992

Abstract: Nuclear magnetic resonance studies of the complex between the consensus zinc finger peptide CP-1 and the
paramagnetic ion Co(I1) have allowed the orientation and anisotropy of the magnetic susceptibility tensor to be determined.
Two approaches were used. First, 70 resonances were assigned for the CP-1-Co(I1) complex using a combination of
2-D N M R techniques and characterization of sequence variants. The tensor was determined by maximizing the
agreement between observed and calculated chemical shifts while varying the orientation and anisotropy of the tensor.
The second approach compared the observed and calculated integrated spectra of the Co(I1) complex while optimizing
the tensor parameters. Both methods found the axial and equatorial anisotropies of the tensor to be xax= xZz- 0.5(xXX
+ xvy) = 2280 f 400 VVk and xeq= xxX- xvy = 280 f 310 VVk. The largest value of the magnetic susceptibility
tensor lies approximately along the bisector of one of the sulfur-Co(I1)-nitrogen angles. The knowledge of this tensor
will allow refinement of the three-dimensional structure of the peptide and its complexes with DNA with constraints
not available from traditional N M R studies.

Paramagnetic ions hold great promise as probes in magnetic

resonance studies.'-' Zinc finger peptides represent a natural
system to investigate these effects. These small peptides (25-30
amino acids) bind one Zn(1I) or another similar ion to fold into
a stable structure.a The Zn(I1) complexes of such peptides
have been studied extensively by N M R We report
herein the first detailed N M R studies of a zinc finger peptide
substituted with the paramagnetic ion Co(I1). Using a combi-
nation of 2-D N M R techniques, characterization of sequence
variants, and novel refinement methods, we have been able to
determine the orientation and anisotropy of the magnetic
susceptibility tensor.

Experimental Section
All peptides were synthesized with the use of a Milligen/Biosearch
9050 peptidesynthesizer and purifiedand reduced asdescribed previously.8
All experiments were performed under a n atmosphere of 2-5% hydrogen
16.0 I t .0 6.0 4.0 0.0
balanced with nitrogen to avoid cysteine oxidation. N M R samples were
PPm
prepared with 1.1-1.2 equiv of cobalt(I1) and adjusted to the desired p H
with deuterated Tris (MSD Isotopes). IH N M R data were obtained on
Varian XL400 and Bruker AM600 N M R spectrometers and processed
with software supplied with the spectrometers or with FELIX (Hare
Research, Inc., Woodinville, WA). MagnitudeCOSY9.loandT0CSY1
' Dedicated to Professor Richard H. Holm on the occasion of this 60th
birthday.
( I ) Bertini, 1.; Luchinot,C. NMRofParamagneticMoleculesin Biological
Systems; Benjamin/Cummings Publishing Co., Inc.: Menlo Park, CA. 1986.
(2) Lenkinski, R. E.; Glickson, J. D. In The Peptides; Udenfriend, S.,
Meienhofer, J., Eds.; Academic Press, Inc.: Orlando, FL, 1985; Vol. 7, pp
301-353.
(3) Emerson, S. D.; La Mar, G. N. Biochemistry 1990, 29, 1556.
(4) Berg, J. M. J. B i d . Chem. 1990, 265, 65 13.
(5) Klug, A.; Rhodes, D. Trends Biochem. Sci. 1987, 12, 464.
(6) Kaptein, R. Curr. Op. Struc. Bioi. 1991, 1 , 63; 1992, 2, 109.
(7) (a) Lee, M. S.;Gippert, G. P.; Soman, K. V.; Case, D. A,; Wright, P.
E. Science 1989,245,635. (b) Lee, M. S.; Cavanagh, J.; Wright, P. E. FEBS
Leu. 1989, 254, 159. (c) Parraga, G.;Horvath, S.J.; Eisen, A.; Taylor, W.
E.; Hood, L.; Young, E. T.; Klevit. R. E. Science 1988,241, 1489. (d) Klevit,
R. E.; Herriott, J. R.; Horvath. S. J. Proteins: Sfrucf.,Funct., Genef. 1990,
7, 215. (e) Omichinski, J. G.; Clore, G. M.; Apella, E.; Sakaguchi, K.;
Gronenborn, A. M. Biochemistry 1990,29,9324. (f) Kochoyan, M.; Havel,
T. F.; Nguyen, D. T.; Dahl,C. E.;Keutmann, H.T.; Weiss, M. A. Biochemistry Id. 0 12.0 8.0 4.0 0.0
1991, 30, 3371. (g) Kochoyan, M.; Keutmann, H. T.; Weiss, M. A. PP'
Biochemisfry 1991, 30, 9396. (h) Neuhaus, D.; Nakaseko, Y.; Nagai, K.;
Klug, A. FEBS Leu. 1990, 262, 179. Figure 1. The 600 M H z one-dimensional ' H N M R spectrum of the (A)
(8) Krizek, B. A.; Amann, B.T.; Kilfoil, V. J.; Merkle, D. L.; Berg, J. M.
J . Am. Chem.Soc. 1991, 113.4518. Zn(1l) and (B) Co(I1) complexes of CP-1 in D20,2.1 mM, p H 7.20 and
(9) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976,64, 2229. 7.05, respectively, a t 25 OC. Additional resonances shifted further
(IO)Nagayama, K.; Kumar, A.; Wuthrich, K.; Ernst, R. R. J. Magn. downfieldduetovery largedipolarand/orcontactshifts have beendetected
Reson. 1980, 40, 321. but a r e not shown.

OOO2-7863/93/
1515-2577$04.O0/0 0 1993 American Chemical Society
2578 J. Am. Chem. SOC.,Vol. 115, No. 7, 1993 Harper et al.

3 4 1

2-i
m

7 O
QQ O Q Y’

0 0 e P B
i o Ea“

2 1 2 3 2 1 2 3
Figure 2. A portion of the 400-MHz magnitude COSY spectrum of the Co(l1) complex of (A) CP-I,2.1 mM, pH 7.05 and (a) CP(K3A), 6.78 mM,
pH 6.55 in D20, at 25 O C . Assignments for the appropriate resonances are indicated.

Table I. Observed versus Calculated Shifts

Znob COobs (LW/H)diP Cocalc Znob Cook (LW/H)diP COcalc
IPro a 4.27 3.87 -0.33 3.94 I4Lys N 8.76 8.44 -0.30 8.46
‘Pro B 2.31 1.85 -0.39 1.92 ‘4Lys a 3.07 2.73 -0.35 2.72
‘Pro 8’ 1.85 1.27 -0.52 1.33 ‘4Lys B 1.54 1.40 -0.16 1.38
‘Pro y 1.98 1.64 -0.29 1.69 ‘4Lys 8’ 1.13 0.87 -0.17 0.96
I Pro y’ 1.91 1.48 -0.37 1.54 I5Ser N 8.71 8.42 -0.34 8.37
‘Pro 8 3.34 3.05 -0.27 3.07 ISSer a 3.92 3.74 -0.30 3.62
2Tyr N 8.99 8.21 -0.62 8.37 ISSer 3.86 3.61 -0.35 3.51
2Tyr a 4.66 3.97 -0.64 4.02 15Ser 8’ 3.81 3.66 -0.26 3.55
2Tyr B 3.10 2.44 -0.77 2.33 I6Asp N 6.86 6.38 -0.66 6.20
*Tyr 8’ 2.72 1.90 -0.96 1.76 I6Asp a 4.39 3.80 -1.03 3.36
2Tyr 8 7.17 6.67 -0.43 6.63 I6Asp @ 2.98 2.09 -1.47 1.51
*Tyr c 6.86 6.75 -0.16 6.78 I6Asp 8’ 2.77 2.34 -0.88 1.89
)Lys N 8.81 7.67 -1.06 7.75 I7Leu N 7.08 6.25 -0.86 6.22
3Lys a 5.08 2.04 -3.17 1.91 17Leua 3.24 1.04 -1.61 1.63
3LYS B 1.59 -0.34 -2.36 -0.77 I7Leu 2.08 1.54 -0.54 1.54
3Lys 8’ 1.44 -0.01 -1.60 -0.16 I7Leu 8’ 1.26 -0.03 -1.40 -0.14
’LYS Y 1.17 -1.19 -0.96 0.21 ‘?Leu y 1.63 1.77 0.47 2.10
3Lys y’ 1.17 -1.52 -1.03 0.14 I7Leu 8 0.99 0.83 1.02 2.01
loser N 8.03 7.63 0.50 8.53 InVal N 8.19 7.70 -0.38 7.8 I
“JSer a 5.25 3.07 -1.02 4.23 ‘Val a 3.66 3.91 0.39 4.05
loser 3.50 2.65 -0.28 3.22 ‘ValB 1.98 1.83 -0.12 1.86
“Phe N 8.80 7.41 -1.19 7.61 ’Valy 0.98 1.12 0.15 1.13
“Phe a 4.75 4.18 -1.01 3.74 ’Wal y’ 0.89 0.96 0.12 1.01
“Phe B 3.50 2.71 -1.3 1 2.19 I9Lys N 7.45 6.68 -0.66 6.79
”Phe 8’ 2.67 1.48 -1.41 1.26 ‘9Lys a 3.91 3.18 -0.59 3.32
I?3er N 9.21 8.78 -0.70 8.51 ‘9Lys p 1.84 1.08 -0.97 0.87
12Ser a 4.45 4.14 -0.43 4.02 ‘9Lys 8’ 1.73 0.97 -0.84 0.89
’%er @ 4.05 3.89 -0.29 3.76 ‘9Lys y 1.39 0.70 -0.45 0.94
1%er 8’ 4.05 3.82 -0.32 3.73 ‘9Lys y’ 1.39 0.70 -0.52 0.87
I3Gln N 8.14 7.68 -0.61 7.53 23Thra 4.13 1.72 -2.23 1.90
I3Gln a 4.91 4.62 -0.36 4.55 23Thr@ 4.03 -0.53 -4.23 -0.20
I3Gln 2.23 2.00 -0.32 1.91 23Thry 1.17 -2.63 -3.57 -2.40
I3Gln 8’ 2.00 1.73 -0.43 1.57 26Gly N 8.37 7.70 -0.43 7.94
I3Gln y 2.46 2.25 -0.29 2.17 26Glya 4.02 3.62 -0.47 3.55
I3Gln y’ 2.41 2.19 -0.32 2.09 2bGlya’ 3.96 3.81 -0.07 3.89

experiments were performed on peptide samples with concentrations in G 1 y L y s S e r P h e S e r G 1 n L y s S e r A s p L e u V a 1L y s H i s -

the range of 1 to 7 mM in D20 (Aldrich, lOO.O%), PH 6.5-7.5,298 K. GlnArgThrHisThrGly.8 the one-dimensional IH NMRspectra
Magnitude cosy and NOESY‘3-’5experiments were preformed On ofthe Zn(II) and Co(II) complexes ofthis peptide are shown in
peptide samples in 10%D20/90% H20, pH 5.6-5.8, respectively. The
mixing times for the NOESY and TOCSY experiments were and Figure 1. Note the large shifts of resonances to positions from
21 ms, respectively. The water signal was suppressed by presaturation. -2.64 to +17.3 ppm in the Co(I1) complex spectrum. These
shifts are due to dipolar interactions between protons and the
Results and Discussion
Studies were performed with the concensus zinc finger peptide (12) Bax, A,; Davis, D. G.J. Magn. Reson. 1985. 65. 355.
CP- 1 which has the sequence ProTyrLysCysProGluCys- (13) Jeencr, J.; Meier, B. H.; Bachmann, P.;Ernst, R.R. J . Chem. Phys.
1919. 71, 4546.
( I I ) Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521. (14) Macura, S.;Ernst, R. R. Mol. Phys. 1980, 41, 95.
N M R Studies of a Cobalt-Substituted Zinc Finger Peptide J. Am. Chem. Soc.. Vol. 115, No. 7, 1993 2519

10 '
0
8
A

E
a 6
W

G 4
5
3 2
c
3
0
z o
-2
/-, 1 -12 ' I I I I

-4
I I I I I 0 90 180 270 360
-4 -2 0 2 4 6 8 10 torsional angle (degrees)
observed shift (ppm) Figure 5. Calculated NMR shifts versus torsional angle for 23Thrin the
Co(I1) complex of CP-I. The dotted line is for the y-methyl group and
the solid line the 8-hydrogen. The angle is defined as C,-C&&O.
The horizontal lines denote the respective observed NMR shifts. The
inset shows the combined deviation as a function of torsional angle.

can be characterizedby an effective magneticsusceptibilitytensor.

Attempts to locatesignalsdue to thecontact-shifted metal-binding
residues expected outside this range have been unsuccessful.
Further studies at lower field strengths may be useful in this
regard.
In order to determine the tensor properties, two approaches
were taken. The first method required that a number of proton
resonances be assigned in the Co(I1) complex. The spectrum of
the Zn(I1) complex had been assigned previously.8 Partial
assignment of the Co(I1) was achieved by magnitude COSY
experiments on the Co(I1) complexes of CP-1 and on a series of
peptides for which selected residues were replaced by alanine.
The variants studied included two single variants, CP-1(K3A)
and CP-I(PSS), and two double variants, CP-I(E6A, T23A)
and CP-1(K9A, T25A). Chemical shifts for 3Lysand 23Thrwere
assigned by comparing the COSY peak patterns of the Co(I1)-
Figure 3. (A) The observed versus calculated NMR shifts for the Co(l1) bound variants with those from the CP-l-Co(I1) complex. This
complex of CP-1. (B) An isoshift surface denoting the distance and is illustrated for the CP- 1 (K3A) in Figure 2. Assignments were
orientation necessary for a 1 ppm downfieldor upfield shift. The surface not found for "lu, 9Lys,and 2SThr and the resonances for these
is in the same orientation as the accompanying structure of the zinc residues were assumed to be too broad to appear in the COSY
finger peptide complex. spectra under the conditions used. Although peaks were found
associated with 5Pr0, they have not yet been unambiguously
assigned. A minimalist zinc finger peptide MZF,16 whose
sequence is LysTyrAlaCys(Ala),Cys(Ala),Phe(Ala),Lys
(Ala),&(Ala),His(~),HisAlaLys, proved to beparticularly
useful in that resonances for the hydrophobic core residues,
underlined, were able to be assigned. The shifts for these
resonances are very nearly identical for all of the peptidesstudied,
supporting the conservation of both the three-dimensional
structure and the electronic properties of the Co(I1) ion among
the variant peptides. The resonances for two residues, '3Glu and
V a l , were assigned by observing the similarity of their chemical
shifts and COSY peak patterns to those found in the CP-I-Zn-
(11) complex. Using all of the above assignments as well as the
NHt-NHj+l, aN, @N,and NHt-NH,+3 NOFSY patternsobserved
in HzO samplesof CP-1 complexed with Zn(I1) and with Co(II),
a total of 70 resonances were assigned. These are listed in Table
I.
Using the first method, the tensor was determined by maxi-
20 15 10 5 0 -5 mizing the agreement between the observed and calculated NMR
PPm shifts for the CP-l-Co(I1) complex while varying the anisotropy
Figure 4. The observed (solid line) versus calculated (closed circles) and orientation of the tensor. The calculated NMR shifts were
integrated NMR spectra for the Co(1l) complex of CP-1.
( 1 5) Bodenhausen, G.; Kogler, H.; Ernst, R. R. J . Magn. Reson. 1984.58,
370.
paramagnetic ion. The differences between the chemical shifts (16) Michael, S. F.; Kilfoil, V. J.; Schmidt, M. H.; Amann, B. T.; Berg,
for corresponding resonances in the Co(I1) and Zn(I1) complexes J. M. Proc. Narl. Acad. Sci. U S A . 1992.89, 4196.
2580 J. Am. Chem. Soc., Vol. 115, No. 7, 1993 Harper et ai.

determined by adding the paramagnetic dipolar shift contribution The tensor can be further refined by simultaneously refining
tothepreviouslydetermined NMRshiftsfrom theZn(I1) complex. the structure. The significant error bars on the anisotropy of the
The dipolar shift, (AH/WdiP,was calculated from the equation tensor are largely due to the imprecision of the structure. As in
any NMR-derived structure, the conformations of the solvent
exposed side chains are often not well determined. For example,
the side chain of 23Thr was not well constrained by the NMR
in2 e cos 24 data of the CP-1-Zn(I1) complex alone. The addition of the
constraints due to the dipolar shifts uniquely defined the
where r is the metal-to-proton distance, 8 and 4 are the spherical conformation of this side chain. Only one conformation, where
polar angles with respect to the principal axes of the susceptibility the torsional angle is around 166', can reproduce the observed
tensor, and xXX,xyy, and xzz are the principal magnetic suscep Co(I1) N M R shifts for both the @-hydrogen and the y-methyl
tibilities. The paramagnetic contact shift contribution, a through group, as seen in Figure 5.
bond effect, is expected to be negligible for all residues except
those directly bonded to the metal. Since our assignments did Conclusions
not include any of the metal binding ligands, contact shift effects These results demonstrate the ability to determine the rhombic
were not considered further. The coordinates used were from the magnetic susceptibility tensor for a tetrahedral Co(I1) complex
refined NMRstructureoftheCP-1-Zn(I1) complex.i7 Theaxial of a medium sized peptide using only N M R and structural data.
and equatorial anisotropies of the tensor were found to be Xax = One method involved detailed assignment of a number of
xZr- 0.5(xXX+ xyv) = 2280 f 400 VVk and xCs= xxx- xyy = resonances for the Co(I1) followed by tensor refinement. A second
280 f 3 10 VVk and the principal axes were found to be within method was developed that did not require any assignments for
21° of the bisectors of the ligand-metal-ligand axes. This the Co(I1) complex but used only the integrated spectrum.
orientation is consistent with results on simple tetrahedral Co(I1) Although the second method was not robust enough to allow
complexes.I8 Even though the tensor is nearly axial, the largest simultaneous optimization of all tensor parameters, it did yield
principal susceptibility does not lie along the bisector of the unique a preliminary tensor that provided initial predictions for the shifts
sulfur-Co(I1)-sulfur angle but rather along the bisector of one for the Co(I1) complex. Only a few assignments were necessary
of the sulfur-Co(I1)-nitrogen angles. to resolve the ambiguities from this method. This tensor we have
In the second method, the agreement between the observed determined will be useful for refining the structures of zinc finger
and calculated integrated spectra of the Co(I1) complexes was peptidemetal complexes and for examining the structures of
maximized. Theobserved spectrum was integrated and chemical zinc finger peptide-DNA complexes. Calculations using the
shift values corresponding to lo%, 20%, etc. of the total integrated tensor and the structure of the Zif268-DNA complexi suggested
area were determined. These values were compared with those that shifts up to 2 ppm for certain sugar protons and up to 0.1
calculated assuming particular values for the susceptibility tensor ppm for the imino protons of the DNA can be expected via these
anisotropy. The principal axes of the tensor were assumed to dipolar interactions even at distances of over 20 A.
bisect the ligand-metal-ligand angles. The best agreement was
observed with anisotropies of xax= 2400 VVk and xss = 500 Acknowledgment. Financial support for this work has been
VVk. These results arevery similar to those from the first method provided by theNationa1 Institutesof Health, theNationa1 Science
yet required only a one-dimensional N M R spectrum of the Co- Foundation, and the Lucille P. Markey Trust. The N M R
(11) complex. However, this method did not incorporate enough spectrometers were purchased with assistance from the National
constraints to allow the orientation of the tensor to be refined. Institutes of Health and The National Science Foundation.
Observed and calculated shifts from both methods aresummarized L.V.H. is the recipient of a Howard Hughes Predoctoral
in Figure 3. Fellowship, V.K.V. was the recipient of a scholarship from the
x
(17) Vinson, V. K.; Amann, B. T.; Berg, J. M., manuscript in pre aration.
The root-mean-square deviations for a set of 25 structures are 0.45 for the
backbone atoms and 0.92 A for all atoms.
Fulbright Foundation, B.T.A. was the recipient of a Fellowship
from the Institute for Biophysical Research on Macromolecular
(18) Horrocks, W. D.; Hall, D. D. Coord. Chem. Reo. 1971.6, 147. Assemblies, and J.M.B. is a Presidential Young Investigator and
(19) Pavletich, N. P.;Pabo, C. 0. Science 1991, 252, 809. an Alfred P. Sloan Fellow.