FLUOROTYROSINE ALKALINE PHOSPHATASE I9F NMR
Crook, E. M., Mathias, A. P., and Rabin, B. R. (1960b), Biochem. J. 74, 234. Easley, C. W. ( 1 9 6 9 , Biochim. Biophys. Acta 107, 386. Eldjarn, L., and Pihl, A. ( I 957), J . Biol. Chem. 225, 499. Feitelson, J., and Hayon, E. (1973), Photochem. Photobiol. 17, 265. Gall, W. E., and Edelman, G. M. (1970), Biochemistry 9, 3188. Goldberger, R. F.. Epstein, C. J., and Anfinsen, C. B. (1964), J . Biol. Chem. 239, 1406. Gutcho, M., and Laufer, L. (1954). Glutathione, New York, N . Y., Academic Press, p 79. Harrington, W . F., and Schellman, J. A. (1956), C. R. Trav. Lab. Carisberg, Ser. Chim. 30, 2 1 . Hill, D., and Spivey, 0. (1974), Anal. Biochem. (in press). Hirs, C. H. W., Moore, S., and Stein, W. H. (1960), J . Biol. Chem. 235, 633. Kartha, G., Bello, J., and Harker, D. (1967), Nature (London) 213, 862. Kauzmann, W. (1959), in Symposium on Sulfur in Proteins, 1958, Falmouth, Mass., Benesch, R., Ed., New York, N . Y., Academic Press, p 93.
Longworth, J. W. (1968), Photochem. Photobioi. 7 , 587. Lowry, 0. H., Rosebrough, N. J., Farr, L. A., and Randall R. J. (1951),J. Biol. Chem. 193, 265. Offord, R. E. (1966), Nature (London) 211, 591. Ristow, S., and Wetlaufer, D. B. (1973), Biochem. Biophys. Res. Commun. SO, 544. Saxena, V. P., and Wetlaufer, D. B. (1970), Biochemistry 9, 5015. Sela, M., and Anfinsen, C. B. ( 1 957), Biochim. Biophys. Acta 24, 229. Sela, M., and Lifson, S. ( 1 959), Biochim. Biophys. Acta 36, 471. Spackman, D. H., Stein, W. H., and Moore, S. (1960), J . Biol. Chem. 235, 648. Stevenson, K. J. (1971), Anal. Biochem. 40, 29. White, F. H., Jr. (1961), J . Biol. Chem. 236, 1353. White, F. H., Jr. (1967). Methods Enzymol. 11, 481. Woody, R. W., Friedman, M. E., and Scheraga, H. A. ( 1 966), Biochemistry 5, 2034. Wyckoff, H. W., Hardman, K. D., Allewell, N. M., Tadashi, I., Johnson, L. N., and Richards, F. M. (1967), J . Biol. Chem. 242, 3984.
Fluorotyrosine Alkaline Phosphatase. 19FNuclear Magnetic Resonance Relaxation Times and Molecular Motion of the Individual Fluorotyrosinest William E. Hull* and Brian D. Sykes*
ABSTRACT: Alkaline phosphatase from Escherichia coli has been labeled in vivo with m-fluorotyrosine and the I9F nuclear magnetic resonance (nmr) spectrum of the fully active labeled protein shows 1 1 resolvable resonances corresponding to the 11 known tyrosines per subunit. Nuclear spin relaxation times T I and T2 have been determined for each I9F resonance. Consideration of the theory of dipole-dipole relaxation between unlike spins (I H and I9F) results in the following conclusions. First, the relaxation times are insensitive to internal rotation about the Cp-aromatic ring bond. Secondly, the data require that motion about the C,-Cp bond have a correlation time of 210-6 sec; hence, such motion does not contribute significantly to relaxation. AI1 of the relaxation data are well represented by a
model which assumes (1) isotropic motion of the protein as a whole with a rotational correlation time rc N 70 nsec and (2) a varying degree of ‘‘intermolecular’’ contribution to the I9F relaxation in tyrosine residues by protons on nearby residues. Finally, the ‘‘intermolecular’’ relaxation exhibited a strong correlation with the I9F chemical shift; the contribution of “intermolecular” relaxation was roughly proportional to the shift of a tyrosine from the position of the denatured protein resonance. Thus, I9F nmr is a very useful tool for studying the general tertiary or quaternary structure of a protein, its motional properties, and differences in the local environments of particular residues.
T h e alkaline phosphomonoesterase (EC 3.1.3.1) from Escherichia coli is a metalloprotein consisting of two identical subunits (mol wt 86,00O/dimer) and requires a t least two tightly bound zinc atoms for full activity (Applebury and Coleman, 1969; Reynolds and Schlesinger, 1969; Csopak et ai., 1972).
This enzyme catalyzes the hydrolysis of a wide variety of phosphate esters with very little specificity for substrate. The catalytic mechanism of alkaline phosphatase has been a subject of considerable interest in recent years and it appears that at alkaline pH a slow enzyme conformational change is the ratelimiting step and thus responsible for the uniform rates of substrate hydrolysis (Halford, 1972). Controversial questions of stoichiometry and subunit interaction are also of importance in understanding the mechanism of alkaline phosphatase (Lazdunski et al., 197 I ; Bloch and Schlesinger, 1973). In order to obtain additional specific information concerning the structure-function relationships for alkaline phosphatase, we have undertaken a study of the protein structure using a
From the Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138. Received March 18, 1974. This work was supported by National Institutes of Health Grant GM-17190 (B. D. S . ) . This work was presented at the 15th Experimental Nuclear Magnetic Resonance Conference: Raleigh, N. C.. April 29-May I , 1974. National Science Foundation Predoctoral Fellow, 1973-1 974.
BIOCHEMISTRY,
VOL.
13, NO. 17, 1974
3431
HULL A N D SYKES
fluorotyrosine protein derivative (Sykes et al., 1974). This fully active labeled enzyme is produced by in vivo incorporation of m-fluorotyrosine, and it appears that all 11 tyrosines in the alkaline phosphatase subunit are uniformly replaced by fluorotyrosine. There are several distinct advantages in using I9F nuclear magnetic resonance (nmr) in the study of protein structure. First, of course: I9F is a 10090 abundant spin-half-nucleus which is 83% as sensitive as 'H. Secondly, interference from solvent or buffer resonances is avoided, allowing full utilization of the signal-noise gain capabilities of Fourier transform nmr. Thirdly, use of an I9F label allows one to observe a small number of specific amino acid residues as well-resolved resonances, whereas the IH nmr spectrum of alkaline phosphatase is an uninformative broad envelope. Finally, I9F is considerably more sensitive to local magnetic environment than IH; thus, it is more likely that individual resonances will be resolved, and these resonances will in turn provide a sensitive probe of the conformational state of the protein. In this paper we will discuss I9F relaxation times and chemical shifts for the fluorotyrosine alkaline phosphatase and describe what information these results provide concerning molecular motion and the local environment of the tyrosine residues. Experimental Procedure
Materials. Fluorotyrosine alkaline phosphatase (the generous gift of Dr. H. I. Weingarten which was prepared in the laboratory of Professor Milton J. Schlesinger, Washington University, St. Louis, Mo.) was isolated from a tyrosine auxotroph of E . coli w3747 (ATCC 27256) as previously described (Sykes et al., 1974). The enzyme was received as lyophilized powder after dialysis against distilled water. The specific activity in 1.0 M Tris-1.0 mM p-nitrophenyl phosphate at pH 8, 25", was 3000 f 200 pmol/hr per mg. The specific activity of the pure wild-type enzyme is 3250 pmol/hr per mg (Malamy and Horecker, 1964). Tris( hydroxymethy1)aminomethane was obtained as the LJltra Pure grade from Mann Research Laboratories. All other chemicals were reagent grade. Sample Preparation. All of the relaxation time and chemical shift data presented here were obtained on a single sample of fluorotyrosine alkaline phosphatase containing about 6 mg of lyophilized protein dissolved in 0.25 ml of 0.5 M Tris-D2O buffer. The pH meter reading (Beckman Expandomatic) was 7.83 at 30'. N m r Methods. The 19FFourier transform (FT) nmr spectra were obtained at 94.16 M H z with a Varian XL-100-15 spectrometer equipped with a Varian 620-i 16K computer and a Computer Operations, Inc., L I N C magnetic tape unit. An internal deuterium lock (D20), a 90' pulse of 15 wsec, and spectral width of 2500 Hz were used for all protein spectra. The ambient probe temperature was in the range 25-30', Two types of accumulation techniques were used for the spectra discussed here. For single spectra taken with one set of parameters the Varian long-term averaging or Block mode method was used. A group of transients is collected, an exponential sensitivity enhancement function applied, and the FT spectrum computed. This spectrum is stored and another batch of transients collected, weighted, and transformed. This new spectrum is added to the previous one using double-precision arithmetic. This procedure is continued always adding the new block or spectrum to the sum of all previous blocks. In this way the signal-noise gain is achieved by averaging a small number of spectra rather than a very large number of transients. The problems inherent with a finite computer word length and the necessity to scale down incoming transient data to prevent ov-
3432
BIOCHEMISTRY,
VOL.
13,
KO
11,
1914
erflow place a finite limit on the number of transients that can be usefully acquired in a single block. Hence, for applications involving 50,000-100,000 transients we have obtained improved spectra by using the Block averaging technique. For 7 ' 1 measurements the progressive saturation or (90 - T ) , , method was used (Freeman and Hill, 1971). This involves taking a series of spectra all with 90' pulses but with different pulse intervals T. As described below the values of TI for each resonance can be derived from the dependence of the observed magnetization on the value of T. Since five spectra require 40-50 hr of accumulation time it is necessary that systematic error due to long-term fluctuation in instrument gain or other variables be avoided. This is accomplished by using a modified Fourier transform computer program which acquires a specific number of transients a t one T value, stores the resultant freeinduction decay (FID) on tape, acquires a batch of transients at another 7 value, and stores this FID on tape. This procedure continues until all the T values have been used and then the cycle is started again. At each point where a new T value is selected the previously collected FID for that 7 value is loaded from tape into core and accumulation resumed as though there were no interruptions. All of the information concerning scaling of the data is retained so that absolute intensities are preserved. By cycling through the series of 7 values several times during the experiment one averages out any instrumental fluctuations. Proton decoupling could not be used because of the negative nuclear Overhauser enhancement (NOE) observed with a protein of this size (Sykes et al., 1974). A chemical-shift scale was obtained by inserting a capillary of trifluoroacetic acid (CF3COOH) into the sample and taking a one-pulse spectrum to obtain the frequency of the CF3COOH resonance. The longterm spectrum of the sample was acquired with no capillary present. Relaxation Time Data Analysis. For progressive saturation the magnetization (peak intensity) is given by N ( T ) = .~i,( --l exp(-T/Ti)) (1) where Mo is the thermal equilibrium magnetization and T I is the spin-lattice relaxation time. It is important to remember that the progressive saturation technique is valid only if T >> Tz*, the decay time for magnetization in the x y plane. This condition is easily met for large proteins such as alkaline phosphatase. Equation 1 assumes that a true and accurate base line can be found from which to measure peak intensities. However, in poorly resolved overlapping spectra this is not possible and one must resort to a more general equation
where the constant B represents the difference between the true and the experimental or constructed base line. In practice an attempt is made to construct a consistent set of base lines for all the spectra in a T I series. In other words, eq 2 allows for the use of an arbitrary base line as long as such a base line is constructed in an identical way for each T value, ;.e., B is independent of T. Three-parameter fits ( B , Mo. T I )of the M vs. T data are obtained using eq 2 and a generalized nonlinear leastsquares analysis. In situations where the experimental base line is expected to be accurate one finds that B N 0 and the value of T I is identical (within the standard deviation) with the value obtained using eq I . For those cases where B is significantly different from zero, then eq 1 and 2 generally give different values for Ti with eq 2 giving a more consistent fit to the data and a lower standard deviation for T I . In this case the T I obtained from eq 2 is the preferred value. Due to the limited number of data points arid modest signal-noise ratio, the stan-
FLUOROTYROSINE
A L K A L I N E PHOSPHATASE
I9F N M R
dard deviation of the T I values generally ranges from 10 to
30%. The values of spin-spin relaxation times T2 were obtained from line-width measurements or estimates. A calibration chart giving the true resonance line width as a function of the observed line width was prepared using the computer program SIMEQ (C. W. Kart and P.J. Vander Haak, private communication) to simulate the fluorotyrosine spectrum with various line widths. This procedure allows one to remove the contribution to the line width from the lH-19F coupling. The line width is also corrected for the contribution from the sensitivity enhancement function. The corrected line width is used to calculate T2 by the relation Tz = 1/(7rAv). Dipole-Dipole Relaxation Theory
In the following discussion of I9F relaxation times we will restrict ourselves to consideration only of dipole-dipole relaxation of I9F by IH. It will be assumed that interaction between fluorines on different tyrosine residues is not important and that relaxation due to dissolved oxygen is not significant (this is generally valid for TI < 1 sec). Finally, since experiments were performed in DzO, the effect of solvent need not be considered and hence the data will be analyzed as relaxation due to rotational diffusion. Relaxation times for a spin i (19F) being relaxed by unlike s p i n s j (IH) may be expressed (Doddrell et al., 1972) as shown in eq 3-7, where y , = 2.5176 X IO4 radian sec-I G-I, w 1 = ( 2 ~ ) ( 9 4 . 1 6MHz), 7,= 2.6753 X l o 4 radian sec-' G-I, wJ = (27r)( 100.06 MHz), and rIJ is the internuclear vector.
F, = J(wj -
~
i
F2 = F1
+) 3 J ( ~ i +) +
4J(O)
6J(wi
+ 6J(wj)
+
wj)
-5 -12
-10
-11
-9
-8
-7
-6
-5
rc
IOQ
FIGURE 1: I9F-'H dipole-dipole relaxation at 94.16 MHz. The solid curves represent T I and Tz for a single I9F nucleus relaxed by 'H nuclei as a function of T ~ the , overall molecular correlation time, using eq 6, 7, 8, 10, and 11. The broken curves represent relaxation times when internal rotation (stochastic diffusion) at an angle of 109' is present with the values of 7j labeling each curve. Equation 9 replaces 8 in the calculations.
length of the vectors is not time dependent. When the angle 0 for internal rotation is zero, the spectral density (eq 9) reduces to eq 8, i.e., isotropic motion. It should be noted that, in the absence of simultaneous irradiation of the IH spins, the I9F spin-lattice relaxation may be more complex than a single exponential with the time constant Tli given in eq 3. In the Discussion section the possible discrepancy between the observed T I and the simple theory will be discussed. No similar discrepancy dependent upon the method of measurement need be considered for T2 (Abragam, 1961). The fluorotyrosine residue has the structure shown below with two of the possible degrees of internal rotation. For the
(6)
(7)
The spectral density functions J are given by eq 8 for isotropic motion -NH-CcvH-CO-
and eq 9 for isotropic motion with internal rotation (Woessner, 1962)
+
+
where 1 / 7 2 = l/r, 1 / q , 1 / 7 3 = 117, 4/7i (for stochastic diffusion), 1 / 7 3 = 1/72 (for 120' jump model, jumping rate = 1/(37i) to either of the two adjacent positions), A = (1/4)(3L2 - I)*, B = 3Lz(1 - L2),C = (3/4)(L2 L = cos 8, and 8 = angle between ri, and the axis of internal rotation. The correlation time 7c represents isotropic tumbling of the molecule as a whole, while 7i represents internal rotation about an axis which itself reorients with correlation time 7,. It is important to keep in mind that eq 3 and 4 are written in a way which tacitly assumes that all of the rij vectors involved in the relaxation reorient with the same correlation times and that the
principal relaxation vector r,, shown, it is clear that for rotation about the aromatic-Cg bond 8 II 0' and hence there would be no significant effect on the relaxation from this motion. However, rotation about the C,-Cg bond has 8 = 109O and this motion could affect the relaxation. Contribution from riJ vectors to other protons on the ring would be 15% because of the sixthpower dependence on distance. Equations 3 and 4 can be rearranged to give
TI
x
~
i= ~ l / ( K-F I )~
(10)
~
i= ~ 2/(KF2) - ~
(11)
j
T2
x j
and these functions are plotted in Figure 1 as a function of iC, both for the isotropic case and for the situation where there is internal rotation (stochastic diffusion) with 8 = 109' and selected values of 7i. Considering the isotropic motion data, it is BIOCHEMISTRY, VOL.
13, NO. 17, 1974
3433
HULL A Y D SYKES
F I G U R E 3:
l
,
-12
-11
J
8
-IO
_-
i 8
-9
!
v8
!
-7
#
!
-6
-5
log Tc
I9F-'H dipole-dipole relaxation at 94.16 MHz. The solid curve represents the value of T I / K T=~ T I i l u for isotropic motion as a function of T ~ The . broken curves illustrate the effect of internal rotation a t a n angle of 109' for the labeled values of 7 , . The calculations a r e identical with those for Figure I . F I C L R E 2:
clear that T I = T2 for T~ 5 sec. When i cN a dispersion of TI and TZ occurs due to the terms J ( w i ) and J(o, L%,) in eq 6. However, since the spectral density term J ( w , - w,) is present the values of TI do not increase with increasing i C> (as would be the case for IH) but continue to decrease sec. For i Cgreater than this value T I inuntil rC N 3 X creases again. The values of T2 decrease monotonically with 7 C since Tz is dominated by J ( 0 ) in eq 7 . When internal rotation is present the shape of the Tl-Tz curves can be significantly altered. Starting with very slow motion ( 7 , = one finds that T I no longer increases as rapidly for i Cgreater than 3 X In the intermediate range where ~i = 10-8-10-9the value of TI becomes almost independent of T~ in the range 10-9-10-6.Finally, for fast rotation, T ] 5 IO-". the T1-T. curves become parallel to the isotropic mo~ are greater by a factor of -8. tion case but T I - T values If one takes the ratio of T I and Tz then the dependence will cancel and one has a function which depends only on 7 , and T , (eq 12). This relationship is presented in Figure 2. One
+
Tl/;iT2 = T ~ A V= F ? / ( 2 ; r F 1 )
(12)
observes that the T I / Tratio ~ a t any i Cis a maximum for isotroi pic motion (5, = a) or when internal rotation is very fast ( ~ < For intermediate values of i i , the TI /Tz ratio can be considerably reduced from its isotropic value and this effect is most dramatic for i i = 10-7-10-8. Experimental values of T I and Tz a t a single frequency are not sufficient to determine unambiguously the terms Zri,-6, 7 c , and T,. However, knowledge of TI, Tz, and i C would allow one to obtain two possible values of ~i from the relationship shown i n Figure 2. If %-i,-h is also known then an unambiguous value of 7i can be obtained. Under conditions where internal rotation is known not to contribute then the T I / T 2ratio gives 7c directly and Zr,,-6 can be calculated from the absolute values of T I and T?. A complete determination of Zrl,-6, T,. and T~ requires measurements of T I and Tz a t more than one nmr frequency. Results and Discussion The I9F nmr spectrum of alkaline phosphatase shown in Fig-
3434
BIOCHEMISTRY.
VOL.
13. NO. 17. 1974
53 52 54 56 58 62 6 2 p p m I9F n m r spectrum of native fluorotyrosine alkaline phospha-
tase at 94.16 MHz. The enzyme concentration was approximatel) 25 mg/ml in 0.5 M Tris-D20 buffer (pH 7.85). 25-30', This represents a concentration of about 0.6 m M for each fluorotyrosine. The chemical shift scale is in parts per million upfield of a capillar) of trifluoroacetic acid. This Fourier transform spectrum was obtained w i t h a spectral width of 2500 Hz, acquisition time of 0.2 sec, and a pulse delay of 0.2 sec. The number of transients per block was 128 and 520 blocks were averaged with a sensitivity enhancement time constant of 0.1 sec. Total time was about 7.5 h r . ure 3 was used to obtain chemical shift and line-width data.' There are clearly 11 distinct resonances matching the number of tyrosines determined from sequence work ( M . J . Schlesinger, private communication). Each tyrosine thus has a unique environment and the chemical-shift range of 1 1 ppm dramatically demonstrates the potential for I9F to monitor small changes in local environment as well as overall protein conformational properties. The chemical-shift difference between pH 7.2 and 10.4 for denatured protein in 6 M guanidine was onl? 0.6 ppm. Hence, the ionization state of a tyrosine has ;i small effect on chemical shift relative to tertiary and/or yuaternarq structure. Although assignment of resonances to specific tyrosines must await more detailed sequence, X-ray, and nmr data. the following general observations can be made. The "F resonances appear to fall into three groups. The four resonances 7-10 are clustered about the position of the denatured randomcoil protein (-58 ppm) and thus appear to reflect surface-like environments exposed to solvent. I n conjunction with this finding it is interesting to note that ultraviolet (uv) spectra indicate that 5 f 1 tyrosines per dimer are exposed to solvent (Reynolds and Schlesinger, 1967). This would suggest that three of the resonances in this group belong to three exposed tqrosines p r subunit (perhaps 7, 8, 9). These three resonances are also sensitive to pH and buffer composition. The second group of resonances labeled 1-6 shows significant perturbations from an "exposed" environment. Kotice the monotonic increase in line width as the chemical shifts move downfield relative to the denatured position. This region ma! represent tyrosines which are buried further ,hithin the hydrophobic body of the protein and hence experience more nearneighbor interaction and/or restriction of internal motion rcsulting in shorter T2 values and greater line widths. The resonance labeled I 1 is quite unique in that i t exhibits a large upfield shift relative to denatured protein. Furthermore. as will be shown in future publications this tyrosine is insensitive to pH. metal binding, or substrate binding. The general clean appearance of the spectrum suggests that the subunits are virtually identical. a t least in terms of the tyrosine environments.
'
The authors apologize for reversing the numbering of resonanccs that was used in a previous publication (Sykes et a / . . 1974).
FLUOROTYROSINE ALKALINE PHOSPHATASE
”F
NMR
i
lot
t
-I 1
1 I 2
02
04 0 6 08
IO
12
14
T (sec )
i
L
50 52 54 56 58 60 62ppm
F I G U R E 4: I9F n m r spectra of native fluorotyrosine alkaline phosphatase at 94.16 MHz, progressive saturation (90 - r),, sequence. A spectral width of 2500 Hz was used with acquisition time 0.15 sec. The pulse delay was varied to give the total time ( 7 ) between pulses which labels each spectrum. The automated T I procedure is described in the Experimental Procedure section and the final FID’s contained -57,000 transients. Fourier transformation was performed after sensitivity enhancement with a time constant of 0.06 sec. Total time was 44 hr.
The spectra presented in Figure 4 provided the data for determination of T I relaxation times for each tyrosine. The absolute peak intensities were analyzed as described in the Experimental Procedure section and the relaxation curve obtained for peak 10 is shown in Figure 5. All of the data concerning chemical shifts and relaxation times are summarized in Table I. If one assumes that the fluorine on tyrosine is relaxed “intramolecularly,” i.e., only by protons on that same residue, then 295% of the relaxation is due to the nearest proton with an internuclear vector of 2.6 A (calculated from the covalent radii used in space-filling Corey-Pauling-Koltun (CPK) models). Using this distance to compute r,,-6 one can obtain expected T I values from Figure 1. The absolute minimum T I would thus be 0.45 sec for isotropic motion without internal rotation. Comparing this value with the data in Table I, it is clear that all resonances except no. 10 have T I values significantly less than the minimum predicted. Since internal rotation can only lead to a higher value of the minimum T I ,there must therefore be a significant ‘‘intermolecular’’ contribution to the ‘9F relaxation from other internuclear vectors involving nearby residues in the tertiary structure of the protein. The “intramolecular” internuclear vectors in aromatic residues are indeed rather long and one would in fact expect some “intermolecular” contribution to relaxation in a folded protein structure. Considering now the ratio Tl/aT2. the data in Table I show that resonances 2-10 have ratios in the range 3.2-7.8. Resonances 1 and 1 1 could not be very accurately determined but probably have ratios of -7. Since the ratio is independent of rv, the variation in the values of this ratio might be due to different degrees of internal rotation for the various tyrosines (Figure 2). An estimate of the molecular correlation time T~ for alkaline phosphatase can be obtained from extrapolation of experimental data for a number of globular proteins as studied by fluorescence depolarization (Yguerabide et al., 1970). These results give a value of T~ for alkaline phosphatase a t 27’ in DzO of 67 nsec. With this value of 7c and Figure 2 one obtains an expected T l / s T 2 of 4.3 for isotropic motion which agrees very well with the experimental values for several resonances. From Figure 2 one observes that the range of experimental ratios could be produced by internal rotation with T~ 5 5 X 1O-Io
FIGURE 5: I9F progressive saturation T I determination for fluorotyrosine alkaline phosphatase. The experimental intensities for peak 10 from Figure 4 are plotted along with the least-squares fitted curve.
or
sec. The first situation (fast rotation) can be ruled out on the basis of Figure 1 since improbably large Zri,-6 values would be required, which is also inconsistent with rapid motion. The slow rotation situation would not be significantly different from simple isotropic motion for T~ 5 lO-’sec. Thus, the experimental data require that internal rotation be very slow about the C,-Co bond, and we will therefore assume that all of the I9F relaxation data can be treated using the simple isotropic motion formalism. The variation of T l / a T 2 most likely represents the uncertainty of the experimental relaxation times. The average value of this ratio for tyrosines 2-10 is 5.25 ~
~~
~
Chemical Shifts and Relaxation Times for Fluorotyrosine Alkaline Phosphatase.a
TABLE I:
Resonance 6CFaCooH
1 2 3 4 5 6 7
8 9 10
11 Denaturedb p H 7.2 p H 10.4 Amino acidc PH 7 pH 12
50.48 52.41 53.73 54.53 54.94 55.48 57.25 57.57 58.07 58.92 61.47
TI (sec)
0.1-0.2 0.12 i 0.03 0.1-0.2 0 . 1 6 =k 0.08 0.21 0.02 0 . 2 4 =k 0 . 0 4 0 . 2 0 i 0.04 0 . 2 8 =k 0.05 0 . 3 9 rt 0 . 0 3 0.46 i 0.07 0.36 i0.1
57.59 0 . 2 2 58.19
=k
0.02
T2 (msec)
4.5 6.1 9.4 512 -16 -16 -16 12 -16 45 11
T,/sT? 7-14 6.2 3.4-7 24.3 4.2 4.8 4.0 7.6 7.8 3.2 7-13
19calcd 21 obsd
3.7 3.4
57.98 3 . 5 i 0 . 3 58.84
Chemical shifts (in parts per million upfield from external trifluoroacetic acid) and T 2 values were obtained from the spectrum i n Figure 3. TI values are from spectra in Figure 4. All data are a t ambient temperatures of 25-30’. Fluorotyrosine alkaline phosphatase -6 mg/ml in 6 M guanidineD20. T I is from a n undecoupled series of spectra, observed TZfrom a ‘H-decoupled spectrum, calculated Tz from a n undecoupled spectrum. Chemical shifts are from a sample containing 6 mM m-fluorotyrosine i n 10 mM Tris-0.1 N NaClD20. TIis from a sample containing 17.6 mM m-fluorotyrosine i n 0.5 M Tris-D20 a t p H 9.5.
’
BIOCHEMISTRY, VOL.
13, NO. 17, 1974
3435
HULL AND SYKES (A) 2 6 2 8 30 3 2 3 4
linter
16 18 2 0 2 2
I ” ’
’
24
’
L A . , 01
02
’
’
I
1
03
04
” ’
108
05
I
I
1
06
07
I
TI (secl
F I G U R E 6: I9F relaxation data for fluorotyrosine alkaline phosphatase. The data from Table I are plotted on a grid with T I as the x coordinate and Au = I / r T z as they coordinate. The boxes about each point represent liberal uncertainty estimates. The solid curve represents the calculated locus of ( T I ,.io) points as a function of rlnlerwhere Zri,-6 = 1/ rlnIra6 + I/rlnIer6,rlnfra= 2.6 A, and rc = 71 nsec.
which gives ic = 71 nsec, agreeing quite well with the value of 67 nsec extrapolated from fluorescence data. From the previous discussion it is clear that there must be a significant “intermolecular” contribution to the I9F relaxation: ;.e..: contribution from protons on other residues. If one expresses Xrij-6 as l/rintra6 1/rinter6,then for a given iC one can calculate T I and T2 as a function of rinter.This procedure has been carried out for T~ = 71 sec and rintra= 2.6 8, and the results are shown as the solid line in Figure 6. Also shown on the ( T I ,Au) axis system of Figure 6 are the points corresponding to the observed relaxation times for each resonance in alkaline phosphatase. The data do indeed show a pattern which is quite consistent with our assumption that the relaxation is govN 70 nsec, and the erned by isotropic motion with a single iC values of rinterrequired to explain the observed relaxation times fall in the very reasonable range of 2.0-3.0 A. Note also the interesting fact that the required values of rintercorrelate very well with the chemical shift of the I9F resonances. The lower field peaks correspond to the more buried tyrosines with the smaller values of rinter,i.e.: more interaction with neighboring residues. Note that rinteris the theoretical value required for relaxation by one additional proton. The actual physical interaction may in fact involve two or more protons at corresponding/J’ greater distances. It should be pointed out that tyrosine 1 1 shows the most deviation from the theoretical curve in Figure 6. Currently, we have no particular explanation for this result but wish to point out that the 71 value was not well determined and this particular tyrosine has consistently demonstrated its unique character in many other experiments. The above model for the relaxation, while completely consistent with the data, is oversimplified and the following points need to be considered: ( I ) the presence of internal motions involving modulation of the internuclear vectors rii, (2) the presence of other relaxation mechanisms, specifically spin rotation and anisotropic chemical shift, and ( 3 ) the possible discrepancy between a measured T I and T I , .N o adequate theory exists a t present to quantitatively treat the problem of internal motions which modulate the internuclear vectors. For the purposes of this discussion, however, we are concerned only with the magnitude of the “intermolecular” interaction between the I9F spin and the ‘ H spins on neighboring residues. Thus, even if the rlntervector obtained from Figure 6 represents an effective dis-
+
3436
BIOCHEMISTRY, VOL.
13, NO. 17, 1974
tance involving modulation, the conclusion that the more shifted residues experience a greater interaction with neighboring residues is unaffected. With respect to other relaxation mechanisms, spin rotation is unimportant for T~ values greater than 10-lo sec. Theoretical estimates of the contribution of chemical-shift anisotropy indicate that it contributes little to T I (this is verified experimentally by the complete negative NOE (Sykes et al., 1974)) but that it could contribute significantly to T2. However, a large contribution would result in a T I / T ~ratio significantly different from the ratio observed experimentally, so that the role of chemical-shift anisotropy is judged to be small a t 23 kG. This question is a t present being investigated experimentally a t higher field strengths. The question of the correspondence between the observed TI and T I ,is not easy to settle in general for these multispin systems. In the limit of two isolated spins (one I9F and one IH), the observed I9F T I for the protein under the conditions of the measurement would be a factor of 2 too short: i.e.: l/Tl(obsd) = 2(1/Tl,). On the other hand, for a model where the I9F spin is in a lattice of equidistant ‘H spins, the observed T I can be shown to be equal to T Ifor , a large number of spins. To the extent that the latter is a more reasonable model for the IH spins of the protein, we have assumed that the measured T I is given by T I , .The error inherent in this assumption is at most a factor of 2 and implies that the “intermolecular” contribution to I9F relaxation may be somewhat overestimated. Table I also includes data for the denatured protein and the free amino acid m-fluorotyrosine. The long T I for the free tyrosine may contain a contribution from dissolved oxygen and from solvent protons. W e are confident, however, that these effects do not significantly influence I9F relaxation times in the native protein. The relaxation times for the random coil denatured protein are also difficult to analyze. The solvent was 6 M guanidine-D20 which would contribute to the relaxation. Also the motional properties of a random coil protein are quite complicated (segmental motion and internal rotation are highly anisotropic) and are not adequately described by current relaxation formalisms. In conclusion, it appears that I9F nmr of a fluoro-labeled protein is a uniquely effective technique for studying questions concerning protein conformation, local environments of particular residues, and motional properties. W e have been able to resolve distinct I9F resonances for each of the 11 tyrosines in the alkaline phosphatase subunit. Relaxation time data demonstrate that these tyrosines do not exhibit significant motional freedom about the C,-Cp bond, while the data are insensitive to motion about the Cg-aromatic bond. The lack of internal rotation about the C,-Cp bond is certainly reasonable since a rather large volume element would be required for such motion. The relaxation data could be very adequately described by isotropic tumbling of the protein molecule with a iC of -70 nsec with a significant contribution to I9F relaxation from nearby amino acid residues. This “intermolecular” contribution to relaxation shows a definite correlation with the I9F chemical shifts. Residues with chemical shifts further away from the denatured protein shift exhibited increased “intermolecular” relaxation. This contribution ranged from -86% for tyrosine 1 to 510% for tyrosine 10. Thus, the unique environment of each tyrosine is clearly reflected in both its chemical shift and relaxation times. I n future publications we will discuss the effects of pH, inorganic phosphate binding, and metal binding on the I9F nmr spectrum and conformational properties of alkaline phosphatase.
BINDING OF
i3co
TO H E M O G L O B I N S
Acknowledgments The authors wish to thank H. I. Weingarten and M. J. Schlesinger for their most generous gift of fluoroenzyme (supported by National Science Foundation Grant GB-27613), and they wish to acknowledge National Science Foundation Grant GP32317 for the purchase of the nmr spectrometer, Steven L. Patt for the automated T I programming, S. E. Halford for helpful discussions and encouragement, and S. H. Smallcombe for helpful criticisms. References Abragam, A. ( 1 961), Principles of Nuclear Magnetism, London, Oxford University Press. Applebury, M. L., and Coleman, J. E. (1969), J . Biol. Chem. 244, 308. Bloch, W., and Schlesinger, M. J. (1973), J. Biol. Chem. 248: 5794. Csopak, H., Falk, K. E., and Szajn, H. (1972), Biochim. Bio-
phys. Acta 258, 466. Doddrell, D., Glushko, V., and Allerhand, A . (1972), J . Chem. Phys. 56, 3683. . Freeman, R., and Hill, H. D. W. (1971), J . Chem. Phys. 54, 3367. Halford, S . E. (1972), Biochem. J . 126, 727. Lazdunski, M., Petitclerc, C., Chappelet, D., and Lazdunski, C. (1971), Eur. J. Biochem. 20, 124. Malamy, M. H., and Horecker, B. L. (1964), Biochemistry 3, 1893. Reynolds, J. A., and Schlesinger, M. J. (1967), Biochemistry 6, 3552. Reynolds, J . A., and Schlesinger, M. J. (1969), Biochemistry 8, 588. Sykes, B. D., Weingarten, H. I., and Schlesinger, M. J. (1974), Proc. Nut. Acad. Sci. U. S. 71, 469. Woessner, D. E. ( 1 962), J . Chem. Phys. 36, 1. Yguerabide, J., Epstein, H. F., and Stryer, L. (1970), J . Mol. Biol. 51, 573.
13CMagnetic Resonance Studies of the Binding of Carbon Monoxide to Various Hemoglobins? Richard B. Moon and John H. Richards*
ABSTRACT: Carbon monoxide binding to hemoglobins from a variety of sources has been studied by 13C nuclear magnetic resonance. The two resonances have been specifically assigned to I3CO bound to a or to @ subunits. The reason for the anomalous shift of 13C0 bound to the a chain of rabbit hemoglobin is
In
discussed with particular reference to residue Phe-48 (CD6). The relative facility with which oxygen displaces carbon monoxide, and the relative thermodynamic affinity for carbon monoxide compared to the unliganded state, of the a and /3 subunits are found to differ.
normal hemoglobin tetramers, the acquisition of a ligand by the iron atom of a particular heme group depends critically on whether or not the other heme groups in the tetrameric molecule are liganded. This dependence of the ligand affinity of one subunit on the existing degree of ligation of other subunits accounts for the positive cooperativity with which most normal hemoglobins bind molecules such as oxygen or carbon monoxide (Antonini and Brunori, 1970). Major conformational differences exist between unliganded deoxyhemoglobin and fully liganded forms of the protein (Muirhead et of., 1967; Perutz et ai., 1968; Muirhead and Greer, 1970; Bolton and Perutz, 1970) but exactly how the ligand affinity of a given heme is modulated by events elsewhere in the molecule is not presently known. Some of the unanswered questions are to what extent these effects may be caused by steric factors operating to exclude ligands from a heme in one protein conformation while in another confirmation providing for unobstructed ligand approach (Perutz, 1970) or to what extent these effects may reflect electronic factors resulting from changes in the interaction between the iron atom of the heme and the proximal histidine (F8) or by changes in the interaction between substituents
about the porphyrin ring (such as the vinyl groups) and the a cloud of the porphyrin. Both families of factors probably contribute and the rigorous dissection into steric or electronic effects undoubtedly oversimplifies the actual situation. For example, the “electronic” effects, though ultimately manifested a t the iron atom, probably have their origin in conformational changes of the polypeptide chain which then influence the proximal histidine. Similarly changes in the orientation of polypeptide chains near the periphery of the porphyrin ring can eventually manifest themselves as “electronic” effects at the iron. To gain insight into some of these questions provides the focus of this work whose ultimate objective is to resolve the origin of the allosteric cooperativity of hemoglobin. In particular we sought to identify the environmental differences experienced by carbon monoxide when bound to the different subunits of various hemoglobins (Moon and Richards, 1972) with some attention to the relative thermodynamic ease of displacement of carbon monoxide by oxygen from cy or @ subunits and the relative affinities of the unliganded subunits for carbon monoxide. We used the I3C nucleus of I3C-enriched carbon monoxide as the probe in this work.
Contribution No. 4774 from the Church Laboratory of Chemical Biology, California Institute of Technology, Pasadena, California 91 109. Received October 16, 1973. Supported by U. S. Public Health ServiceGrants NIHL 15198 and NIHL 15162.
Experimental Section
Materials and Methods. Hemoglobin was prepared from freshly drawn, citrated whole blood of human, bovine, mouse BIOCHEMISTRY, VOL.
13,
NO.
17, 1974
3437
