The utility of the nuclear overhauser effect for peak assignment and structure elucidation in paramagnetic proteins

JOURNAL

OF MAGNETIC

RESONANCE

64,52

l-526 ( 1985)

COMMUNICATIONS The Utility of the Nuclear Overhauser Effect for Peak Assignment and Structure Elucidation in ParamagneticProteins STEPHEN W. UNGER,JULIETTET.J.LECOMTE,ANDGERDN.LAMAR* Department

of Chemistry,

University

of California,

Davis,

California

95616

Received April 8, 1985; revised May 29, 1985

The nuclear Overhauser effect, NOE, represents one ofthe most powerful techniques for peak assignment as well as for solution structure determination in highly folded biopolymers (I, 2). In paramagnetic ions, the presence of unpaired spin(s) is known to diminish the magnitude of the NOE through “paramagnetic leakage” (I, 3). The premise that paramagnetism renders interproton NOES negligible was the basis for approaching the needed peak assignments in ferricytochrome c by first carrying them out via NOES in the reduced diamagnetic protein and then relating the peaks so identified to those of the oxidized protein by saturation transfer via electron exchange (4). Only very recently have data become available that demonstrate NOES can be used for making important assignments in a variety of low-spin ferric hemoproteins (5-8). The surprising magnitude of the NOE in these systems can be rationalized by the ineffective nuclear relaxation by the rapidly relaxing lone iron spin (S = 4). Peak assignments are essential for numerous proteins that are either naturally paramagnetic or exist in interesting paramagnetic derivatives, and for which alternative methods are either incomplete, ineffective, or ambiguous. Thus there are compelling reasons for expanding, if possible, the applicability of NOE measurements to include paramagnetic proteins in general. We address here the question of the scope and utility of the NOE for making assignments in paramagnetic proteins. We select not the optimal case, but a difficult case of a paramagnetic iron protein, namely the high-spin (S = 5) ferric met-aquo myoglobin, metMbH*O. This protein provides an ideal test system since it shows efficient paramagnetic contribution to nuclear relaxation and yet its spectrum is relatively well-resolved by virtue of large hyperline shifts. Although a number of the heme (Fig. 1) resonances have been assigned by isotope labeling (9), the important propionate peaks remain unassigned. We demonstrate that highly useful NOES can be detected in met-aquo myoglobin in spite of the broad lines and efficient spin-lattice nuclear relaxation. NOE data were collected at both 360 and 500 MHz. The downfield hypertine shifted portion of the 500 MHz ‘H NMR spectrum of metMbHz0 in ‘Hz0 at 40°C is illustrated in Fig, 2A; most of the heme side-chain signals are known or expected to resonate * To whom correspondence should be addressed. 521

0022-2364185 $3.00 Copyright 0 1985 by Academic Press Inc. 411rights of reproduction in any form esewd

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I

i

C

d

k FIG. 1. Structure of the heme in met-aquo myoglobin (IO). The carboxyl groups of the propionate side chains have been omitted for clarity. Protons are labeled as in the reference spectrum of Fig. 2.

in this window. Resolved spectral lines are 250-400 Hz wide and isotope labeling has identified peaks u-d as the four heme methyls, i and j as vinyl H,‘s, and k as one of the four propionate HP’s (Fig. 1). As illustrated in Fig. 2B, saturation of a (8CH3) yields a clear NOE (N 1%) to peak e. In the X-ray structure (IO), the closest proton that can experience a large scalar hyperIine shift is the 7-propionate-a-CH (Fig. 1) and we therefore assign e to this proton. Irradiation of e (Fig. 2C) yields the reciprocal NOE to a as well as NOES to h and k. When h is saturated (trace 2D), the reciprocal NOE to e is obtained as is the NOE to peak k. The values for the NOES at 360 MHz are listed in Table 1. The large 7)i-j dictates that e and h are geminal partners (see below) and hence they constitute the 7-propionate a-CH2 proton pair, with e arising from the proton closer to the 8-CH3 (v,,~ = qh-a = 0). The comparable NOES from both e and h to k identify k as the 7-propionate ,&CH as shown in Fig. 1; X-ray structural data confirm comparable distances from the two H,‘s to this H,. Attempts to locate the geminal H;3 partner to k in the diamagnetic region failed because of the enormous intensity of the diamagnetic envelope compared to the very broad hyperfine shifted and paramagnetically relaxed signals. The difference spectra simply could not discriminate the small NOES expected in the region 0 to 10 ppm. Similarly, saturation of peak b (5CH3, Fig. 2E) leads to an NOE to peakfwhich must originate from the 6-propionate-cY-CH (Fig. 1). Saturation off(Fig. 2F) yields only a single but large NOE to peak g, as expected for the geminal cu-CHz group; the reciprocal g tofNOE is also observed (Fig. 2G). Since the only resolved propionate H, (k) can be assigned to the 7-group, the two 6-propionates HP’s must resonate in the diamagnetic region O-10 ppm. The values of the NOES among the 5-CH3 and the 6-propionate peaks b, f; and g are essentially the same as for the 8-CH3 and the 7-propionate peaks a, e, and h. The NOES have led not only to the unambiguous assignments of the four heme propionate H, signals, but also allowed assignments of signals to individual protons in a methylene pair. Isotope labeling would be restricted to at best simply identifying the two H,‘s of a propionate without distinguishing between the diastereotopic pair.

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BG-

c&T

D

L-l l

I 100

I

I

I 80

I

I

1 60

I

I

w 40

20

PPM

FIG.2. Downfield region of the 500 MHz spectrum of met-aquo myoglobin, pH 6.4,4O”C. (A) Reference spectrum recorded with a r/2 observe pulse centered at 60 ppm. (B-G) NOE difference spectra. A 30 ms, 0.05 W decoupler pulse was applied on resonance to achieve 50% saturation and off resonance to collect the reference spectra. A Redfield 2-1-4-I-2 sequence (12) was used as observe pulse with the carrier at -77 ppm (B, C, E) or -48 ppm (D, F, G). A filled circle denotes a peak whose intensity can be accounted for in full by off-resonance saturation. (B) Irradiation of a (8-CH& (C) irradiation of e (7-a); (D) irradiation of h (7-a?; (E) irradiation of b (5-CH,); (F) irradiation ofJ(6-a); (G) irradiation of g (6-a’).

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COMMUNICATIONS TABLE 1 Magnitude of the NOEs in Met-aouo Mvoglobin” ib J

.b

a (8-CHJ) e (7-a) h (7-a')

k (7-8)

a (8-CH,) 1.2 + 0.6 (2.93) 0 (4.38) 0 (4.41)

e (7-a)

h (7-d)

0.4 f 0.2 (2.93)

0 (4.38) 7 -c I(l.77)

4 2

+ 1 (1.77) + 1 (2.45)

3 + I (2.47)

’ 360 MHZ, 40°C pH 6.4 (not corrected for isotope effect). The magnitude is presented as percentage of the corresponding reference peak. ril, the interproton distance in A calculated from the X-ray structure (IO), is given in parentheses. b i is the saturated resonance, j is the observed resonance.

The NOE-based assignments can be further confirmed by measurements of iron-induced nonselective spin-lattice relaxation times, which for the pairs of peaks (a. b), (cf), and (g, h) are at 360 MHz 4.7 + 0.4, 5.2 f 0.5, and 2.4 + 0.4 ms, respectively. Since paramagnetic relaxation varies as r -6 (Fe--H), the relaxation time ordering Tr(e, f) > T,(a, b) > T,(g, h) fits semiquantitatively the predictions based on the Xray crystallographic distances (IO): r(Fe-HH,J ‘u 6.3 A > r(Fe-CH3) - 6.1 A > r(Fe-HHg,h) - 5.9 A. The 7-a-CH2 orientation responsible for the T, differences between H, and Hh places H, at 2.93 A and Hh at 4.38 A from the 8-CH3 group. These distances are consistent with the observation of NOES from a to e and not to h. A similar argument holds for the 6-0+CH2. The comparable NOES obtained from the 7+CH’s to one 7-&CH also agree quantitatively with the X-ray 7-P-CHr orientation (Fig. 1). Thus, the identification of these important heme resonances clearly establishes the potential value of NOES even in highly paramagnetic proteins. Saturation of heme methyl c ( 3-CHs) and d ( 1-CH3) leads to - l-2% NOES to the known vinyl H,‘s (twoproton peak at -7 ppm, not shown). Although both peaks have been identified by isotope labeling, the sizable NOE confirms a cis orientation of the vinyls (vinyls H, pointing toward methyl rather than meso-H). Detectable differences in the two NOES could lead to characterizing slight differences in the mean orientation of the two vinyls. A more quantitative interpretation of the magnitude of observed NOES as well as a more general comprehension of the scope and limitation of their application to paramagnetic proteins result from considering the nature of relaxation in these systems. The NOE in an isolated two-spin system is given by li-j

=

CijlPj

[II

where pj is the intrinsic relaxation rate (inverse of selective Tr) for Hj, and ati is the cross-relaxation rate, which in the pertinent slow-motion case is given by (1)

PI where T, is the rotational correlation

time for the Hi-Hj

vector. For the immobile

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propionate a-CHz (r = 1.77 A), and the known TV- 10 ns (II), we obtain u = - 19 Hz. The selective Tr(p-‘) for a (8-CH3) was determined as 4.7 f 0.5 ms, indistinguishable from the nonselective Tr value. This confirms the complete domination of paramagnetic dipolar relaxation for both selective and nonselective T,‘s. We can thus write pj = TG’(NS), and obtain Ti-j = ~0 T,(NS).

131

In both the 6-a-CH2 and 7-a-CH2 pairs, we estimate qh-e - T,+~ = - 19 (5.2 X 1Oe3) = -0.1 or -10%. We observe -7 + 1% and -7 f 2%, respectively. For g and h, Tl - 2.4 ms, so &.+h - Q-+~= - 19 (2.4 X 10p3) = -0.05 or -5%; observed are 4 & 1% and 5 ~fr2%, respectively. The magnitude of the NOES to be detected is quite small. Therefore, to achieve a satisfactory signal-to-noise ratio, the accumulation of a large number of transients, typically more than 30,000, is needed. However, the large spectral bandwidth (>40 kHz) and the limited digital resolution required (20 Hz/pt) lead to a short acquisition time. As the relaxation is fast, the repetition rate can be rapid and the total experiment time, when observing NOES between hyperfine shifted peaks, is comparable to that for a diamagnetic system. In the present case, we estimate that 77- -0.2% can be detected. Considering a proton Hj with a T, of -5 ms (2 ms, 1 ms), the 0.2% NOE limit in combination with Eqs. [2] and [3] requires that the other proton Hi be within 3.4 A (2.9 A, 2.6 A) assuming no internal motion. For a methylene pair (rii = 1.77 A), t = -0.2% corresponds to a T, of 0.1 ms. In general the detection of a small intensity change becomes more difficult as the line broadens. Hence T2 is often the determining factor in observing the effect; since T2 < T,, this is particularly true for lines with short T,‘s. Practically, there are two other main limitations to the NOE method applied to paramagnetic systems. First, in saturating a line characterized by short Tl and T2, the necessarily strong rf field can induce interfering off-resonance perturbations over a wide region of the spectrum. In that case, true NOES may be difficult to distinguish from off-resonance saturation (spillage). We found that a careful choice of several reference frequencies, comparison of the relative magnitude of the effects under conditions of reduced saturation degree (i.e., 80 and 50%), and the determination of the actual distribution of absorbed power as a function of o&t can, in most cases,alleviate the problem. Second, observation of a small intensity difference on a broad line in the presence of a very large diamagnetic envelope places severe demands on the dynamic range of the receiver and the digitizer. Selective excitation pulse trains such as the Redfield 2-1-4-1-2 sequence (12) are particularly helpful for suppressing the diamagnetic resonances and allowing the full utilization of the dynamic range. In spite of the above limitations, this technique should be of general applicability. Similar studies bearing on hemoglobin derivatives (7, - 40 ns) appear feasible as the T, parameter is essentially independent of the molecular tumbling rate. The method will be especially valuable for the numerous high-spin ferric and ferrous proteins yielding well-resolved hyperfme shifted ‘H NMR spectra and whose heme cannot be substituted without denaturation, consequently for which isotope labeling is precluded (23). Other interesting candidates can be found among the lanthanide derivatives of parvalbumin (14) which often exhibit a well-resolved and relatively sharp spectrum.

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Our present results also suggest that identification of the coordinated ligands (tyrosines and histidines, among others) could be profitably pursued by NOE studies in nonheme iron proteins such as iron-tyrosinate proteins (23, hemerythrin (16) and uteroferrin (I 7). We conclude that the homonuclear NOE has very interesting potential for resonance assignment and solution structure determination in paramagnetic iron proteins. ACKNOWLEDGMENTS This research was supported National Institutes of Health,

by grants HL-16087,

from the National GM-26226.

Science

Foundation,

CHE-84-15329,

and the

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