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[13] Phosphorus-31 nuclear magnetic resonance of phosphoproteins
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
263
information on the distance of a nucleus from the metal, and NOE contains information on the distance between two nuclei, this approach may allow us to redesign the environment of metal ions in proteins, i.e., to design the active cavity of metalloenzymes. This information, coupled with molecular dynamics, is an independent, indispensable approach to the investigation of protein structures. Such studies can also be performed on t3C, J9F, and 3~p nuclei of the protein or of a substrate interacting with a protein. The window of paramagnetic compounds that can be investigated is enlarged by magnetically coupled systems, either natural or artificial. A wealth of information may be obtained on the coupling and on the effects of the coupling on electron relaxation.
[13] P h o s p h o r u s - 3 1 N u c l e a r M a g n e t i c Resonance of Phosphoproteins By HANS J. VOGEL
Introduction The ribosomal protein synthesis machinery utilizes the standard 20 amino acids as the building blocks for proteins. The five elements that are present in these amino acids are hydrogen, carbon, nitrogen, oxygen, and sulfur. Consequently, all proteins can in principle be studied by ill, 13C, |4N/15N, 170, or 33S NMR techniques. Few studies of proteins, however, have utilized 14N, 170, or 33S NMR; the electric quadrupole moment of these nuclei renders resonances for the individual atoms very broad, leading to featureless unresolved spectra.~ Because of their more favorable nuclear properties (spin -- ½), the study of the other three nuclei has provided considerable insight into structural, functional, and dynamic properties of proteins, as will be apparent from the various chapters in this volume. As the element phosphorus is not incorporated during protein biosynthesis, it can only become incorporated into proteins through various posttranslational processes. Consequently only a few phosphorus atoms are found in most phosphoproteins. This has the advantage that the problem of resonance assignment, which can be quite formidable for ~H, ~aC, and ~SN NMR studies of larger proteins, normally does not pose any S. Forsen and B. Lindman, Methods Biochem. Anal. 27, 289 (1981). METHODS IN ENZYMOLOGY, VOL. 177
Copyright © 1989 by Academic Press, lnc. All rights of reproduction in any form reserved.
264
PROTEIN STRUCTURE
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TABLE I PROPERTIES OF SOME COMMON NUCLEI
Nucleus
Resonance frequency (MHz) °
Natural abundance (%)
Sensitivity b
Chemical shift range for biomolecules (ppm)
1H 13C 15N 31p
400.1 100.6 40.5 162.0
99.98 1.11 0.37 100.0
1.00 0.016 0.001 0.066
15 200 100 20
At 9.4 T. b This is for an equal number of nuclei. The receptivity that actually governs the sensitivity of the NMR experiment (and which is usually defined as the isotopic abundance multiplied by the sensitivity) is less for nuclei with low isotopic abundance. a
serious problems with 31p NMR.2 Furthermore, because of the paucity of resonances in 31p N M R spectra of phosphoproteins, it has been possible to pursue studies of proteins with molecular weights up to 500,000. 2 In contrast, the problem of resonance overlap, which is inherent in IH, ~3C, and 15N N M R studies of larger proteins, severely limits the size of protein that can be studied by these three techniques. On the other hand, the information that can be extracted by 3tp NMR is of a different nature than the overall structural detail that can potentially be obtained by the other techniques. In addition to the different way in which phosphorus is incorporated into proteins, it is important to realize that the s~P nucleus has a favorably high resonance frequency and a good sensitivity for NMR experiments (see Table I). It is also 100% naturally abundant, and thus can be studied without the need for introducing isotopic labels. Isotopic labeling can be expensive and experimentally cumbersome, and these factors have limited the use of nuclei with low natural abundance, as in 13C or ~SN NMR. 3 The combination of the biological and physical factors mentioned above explains why 3~p NMR has become such a useful tool for the study of phosphoproteins. In this chapter we will focus on the practical aspects of the technique, which will be illustrated by some selected examples. It is 2 H. J. Vogel, in "Phosphorus-31 NMR: Principles and Applications" (D. G. Gorenstein, ed.), pp. 104-154. Academic Press, Orlando, Florida, 1984. 3 Modern NMR pulse methods can be used to study indirectly tSN or t3C nuclei, by utilizing heteronuclear quantum coherences and detecting the attached protons. These methods provide a great increase in sensitivity, which should overcome to some extent the need for incorporating isotopic labels.
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PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
265
not our intent to give an exhaustive review, and the reader wishing to obtain more details should consult other sources 2,4 or the original literature. Before discussing the experimental aspects of 31p NMR of phosphoproteins, we will briefly review the biological mechanisms through which phosphate can be incorporated into proteins. Phosphorus can become part of a protein through essentially three different mechanisms. The first mechanism is the incorporation of phosphorylated coenzyme into a protein. Such moieties are either extremely rigidly bound to the protein or in some cases they become covalently attached to the side chain of specific amino acids. An obvious example is the coenzyme pyridoxal phosphate, which occurs in the active site of a large number of enzymes. 5 Although the phosphate group of this coenzyme does not appear to be directly involved in enzymatic catalysis, the dephospho (pyridoxal) form generally does not support catalysis. 6,7 As such, the phosphate group presents a nonperturbing spectral reporter group for studying events that take place in the active site of the enzyme. Also, the cofactors FMN and FAD, which are part of many redox proteins, carry phosphate groups that can be studied by 31p NMR. The other two groups of phosphoproteins contain phosphorylated amino acids rather than coenzymes. Of particular interest is a large group of proteins and enzymes whose activity is regulated by reversible enzymatic phosphorylation/dephosphorylation reactions. Specialized protein kinases that can be activated by hormonal stimulation can transfer a phosphoryl group (PO32-) from ATP onto unique serine, threonine, or tyrosine residues on selected target proteins. 8,9 In some nuclear proteins unique histidine, lysine, and arginine side chains can also become phosphorylated. Although these regulatory phosphorylation sites are often far removed from the active site of these enzymes, the phosphorylation has marked effects on the activity. The phosphoryl groups can subsequently be removed by specialized protein phosphatases that are also hormonally controlled. In addition to these regulatory phosphorylations there is a series of food storage proteins in milk and eggs (ovalbumin, casein, and phosvitin) and a series of polyelectrolyte proteins (bone, tooth, and saliva phosphoproteins) that are also phosphorylated by protein kinases. How4 T. L. James, CRC Crit. Rev. Biochem. 18, 1 (1985). 5 K. D. Schnackerz and E. E. Snell, J. Biol. Chem. 258, 4839 (1983). 6 M. E. Mattingly, J. R. Mattingly, and M. Martinez-Carrion, J. Biol. Chem. 257, 8872 (1982). 7 S. G. Withers, N. B. Madsen, B. D. Sykes, M. Takagi, S. Shimomura, and T, Fukui, J. Biol. Chem. 256, 10759 (1981). 8 p. Cohen, Eur. J. Biochem. 151, 439 (1985). 9 A. M. Edelman, D, K. Blumenthal, and E. G. Krebs, Annu. Rev. Biochem. 56, 567 (1987).
266
PROTEIN STRUCTURE
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ever, in this case, the phosphorylation does not play a regulatory role and the proteinous phosphate group is either involved in the binding and precipitation of metal ions or simply functions as a phosphate store. The second group of phosphoproteins, which contain phosphoamino acids, consists of enzymes in which the phosphorylated amino acid is an enzymatic catalytic intermediate.i° The phosphoryl group is transferred from substrates (such as ATP, for example) onto an amino acid side chain in the active site. The residues that become phosphorylated are either a serine (as in the enzymes alkaline phosphatase and phosphoglucomutase), a histidine (as in HPr, succinate-CoA ligase, phosphoglycerate mutase, ATP citrate-lyase), or an aspartate (which has been observed for the Ca 2+-, Na÷,K +-, and the H÷,K+-ATPase). Because they are intermediates in catalysis, their formation is of a transient nature during active turnover. Nevertheless, many of these enzymes can be purified in a stable phosphorylated form that can be studied by 31p NMR. In cases where the covalent phosphoamino acid is unstable, other techniques involving the use of the oxygen isotopes 180 and 170 may be useful to determine whether a phosphorylated intermediate exists 1°,11 and whether it is an obligatory intermediate in catalysis.12 In the following discussion we examine the NMR parameters that can be measured by 31p NMR. We will mainly focus on proteins that contain phosphoamino acids, but some examples involving bound coenzymes will also be mentioned. Sample Preparations For most proteins it will be necessary to make up a 1.5-ml sample with a protein concentration of ->0.2 mM. This sample size is necessary to fill a 10-mm NMR tube to a level that adequately covers the receiver coil and to obtain a signal in a reasonable time (several hours). For proteins that give rise to relatively narrow resonances, it is possible to use protein concentrations that are somewhat lower. The relatively high protein concentration may sometimes result in protein aggregation. In such cases it is advisable to use a larger NMR tube and to reduce the protein concentration while keeping the same amount of protein within the area covered by the receiver coil. For all samples, it is usually advisable to use a Teflon plug, to avoid a vortex in the sample while spinning. Because most phosphorylated compounds carry a negative charge, precautions have to be 10j. R. Knowles,Annu. Rev. Biochem. 49, 877 (1980). ~lj. j. Villafranca, this volume [20]. 12M. J. Wimmerand I. A. Rose, Annu. Rev. Biochem. 47, 1031 (1978).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
267
taken to exclude positively charged paramagnetic metal ions such as Mn 2÷ and Ni 2÷. In order to avoid any such problems, it is therefore advisable to treat all the buffers--and even the 10-20% D20 which is normally used for lock purposes--with a chelating agent such as Chelex 100. It is also of extreme importance to ascertain that the sample does not contain any phosphorylated impurities. For example, nucleic acids or lipids can sometimes be difficult to extract and they can copurify with a protein. The phosphodiester linkages in these molecules give rise to peaks in the 31p NMR spectrum. Extreme conditions, such as organic solvents, are sometimes necessary to remove such impurities. In one case we even were forced to precipitate the protein to prove that a phosphodiester linkage was not part of a protein, but rather a lipid impurity. 13 Not only nucleic acids and lipids can cause problems. For example, the extremely tight binding of the nucleotides AMP or IMP to glycogen phosphorylase necessitates a dialysis procedure against activated charcoal to remove these contaminants. 7 If such precautions are not taken, erroneous results can occur. For example, Fossel et al.14 reported a highly unusual chemical shift for the catalytic phosphoaspartate in the active site of a Na ÷, K ÷ATPase. The enthusiasm for this intriguing observation was severely tempered when subsequent studies showed that the resonance was in fact an ATP contamination and that the real intermediate had a very normal chemical shift. 15Finally, it is important to ascertain that the sample is free from protease and phosphatase activity. As it may be in the spectrometer for several days at temperatures above 4°, it should preferably also be sterile to prevent growth of microorganisms. In some cases addition of a small amount of an antibiotic may be useful. If these conditions are not met, degradation reactions may take place in the course of the experiment, which will generally affect the experimental outcome. Some covalent enzymatic phosphoamino acid intermediates may be intrinsically unstable; this will lead to the gradual disappearance of their resonance and to the appearance of a Pi peak in the spectrum. With respect to the pulsing conditions under which spectra should be collected, it is difficult to generalize. In our experience T~ values for phosphoproteins can vary between 0.25 and 4 sec. Thus the optimal NMR parameters will need to be determined for each individual case. 13 G. D. Armstrong, L. S. Frost, H. J. Vogel, and W. Paranchych, J. Bacteriol. 145, 1167 (1981). 14 E. T. Fossel, R. L. Post, D. S. O'Hara, and T. W. Smith, Biochemistry 20, 7215 (1981). 15 G. M. Sontheimer, H. R. Kalbitzer, and W. Hasselbach, Biochemistry 26, 2701 (1987).
268
PROTEIN STRUCTURE
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TABLE II ACID-BASE STABILITY, PKa VALUES, AND 31p N M R PARAMETERS FOR PHOSPHOAMINO ACIDS a
Phosphoamino acid Phosphomonoesters b Phosphoserine Phosphothreonine Phosphotyrosine Phosphodiesters c RNA, DNA, and phospholipids Acyl phosphates Phosphoaspartate Phosphoramidates NS-Phosphohistidine N1-Phosphohistidine Phospholysine Phosphoarginine
Stable to strong acid
Stable to strong base
+ + +
Chemical shift (ppm) Deprotonated
Protonated
pKa
0.6 0.0 -3.3
5.8 5.9 5.8
0 to - 1.5
No
No
-1.5
-6.5
4.8
-4.5 -5.5 ND -3.0
No No ND -5.4
No No ND 4.3
4.6 4.0 1.0
+
+ + + +
All chemical shifts are reported with reference to 85% H3PO4. Upfield shifts are given a negative sign. ND, Not determined. b The chemical shifts and pH titration behavior of the coenzymes pyridoxal and pyridoxamine phosphate are very similar to those of phosphothreonine, whereas for FMN, these parameters resemble those of phosphoserine. c The diphosphodiester FAD (-10.8/-11.3 ppm) also does not show a titration behavior between pH 3 and 10.
C h e m i c a l Shift a n d N a t u r e of P h o s p h o r y l a t e d A m i n o Acids T h e n a t u r e o f a p h o s p h o a m i n o acid in a n e w l y i s o l a t e d p h o s p h o p r o t e i n is g e n e r a l l y d e t e r m i n e d f r o m a s t u d y of its acid a n d b a s e stability (see T a b l e II). 16-18T h i s s i m p l e test n o r m a l l y p r o v i d e s a r e a s o n a b l e first i n d i c a t i o n b u t s o m e c a u t i o n has to b e e x e r c i s e d , b e c a u s e the r e s u l t is k n o w n to b e d e p e n d e n t o n a d j a c e n t a m i n o acids; m o r e o v e r , e l i m i n a t i o n r e a c t i o n s m a y o c c u r . ~6-1s T h e r e f o r e the n e x t step is u s u a l l y the p u r i f i c a t i o n o f a p h o s p h o p e p t i d e , f r o m w h i c h s u b s e q u e n t l y a p h o s p h o a m i n o acid is obt a i n e d a n d identified. 31p N M R p r o v i d e s a n a l t e r n a t i v e n o n i n v a s i v e m e t h o d for d e t e r m i n i n g the n a t u r e o f p h o s p h o a m i n o acids. 2 T h e c h e m i c a l shifts 19 m e a s u r e d for the v a r i o u s a m i n o acids are quite different (see T a b l e 16G. Taborsky, Adv. Protein Chem. 28, 1 (1974). 17T. M. Mortensen, this series, Vol. 107, p. 1. is j. M. Fujitaki and R. A. Smith, this series, Vol. 107, p. 23. 19Chemical shifts in NMR experiments are always measured with respect to a standard. The commonly used standard for 3~p NMR is 85% H3PO4. This standard is not convenient for
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
269
II) and thus they can be used directly for identification purposes. Naturally, chemical shifts are only a direct monitor of the chemical nature of a phosphoamino acid as long as the incorporation into the protein does not perturb the phosphate moiety. By and large no such deviations have been observed for all the regulatory sites where the phosphoryl group has been introduced by the action of a protein kinase. 2 With the exception of alkaline phosphatase, the majority of the covalent enzymatic intermediates have chemical shifts that are relatively close (<2.0 ppm) to those observed for the free amino acid in solution. Thus the chemical shifts for the histidyl-P of succinate-CoA ligase 2°,2z and ATP citrate-lyase, 22 the aspartyl-P of the ATPases, 15and the serine-P of phosphoglucomutase 23,24were all close to their respective standards. Nevertheless, bond strain has been invoked to explain the 3.8-ppm downfield shift for the serine phosphate residue in the active site of alkaline phosphatase 25,z6 and for the 1.5-ppm downfield shift of the active site Nl-phosphate of histidine in the HPr protein. 27,28Thus it is often advisable to determine the chemical shift for the protein in both the native and a denatured state to be absolutely certain. Denaturation can be brought about by urea, pH extremes, etc. Particularly if one wants to differentiate between the N ~- and N3-phos phohistidine, which have quite similar chemical shifts, this is a necessary s t e p . 27,29 Further confirmation for the nature of the phosphoamino acid may be obtained by looking for a characteristic pH dependence of the shift (see below). Although this step is useful for the identification of phosphomonoesters, unfortunately it does not provide help in identifying phosphohisti-
two reasons. First, it falls right in the middle of the spectrum. Second, and most important, the nature of the standard precludes its use as an internal standard. Although capillaries can be used, the high concentration of the standard also makes this impractical for samples that have weak signals. Therefore, we generally include compounds that have a well-characterized shift, such as phosphocreatine or methylene diphosphonate, as internal standards. The latter is conveniently downfield ( - 2 0 ppm) from the biologically occurring compounds, hut the pH dependence of its chemical shift may cause some problems. 20 H. J. Vogel, W. A. Bridger, and B. D. Sykes, Biochemistry 21, 1126 (1982). 21 H. J. Vogel and W. A. Bridger, J. Biol. Chem. 257, 4834 (1982). 22 S. P. Williams, B. D. Sykes, and W. A. Bridget, Biochemistry 24, 5527 (1985). 23 W. J. Ray, A. S. Mildvan, and J. B. Grutzner, Arch. Biochem. Biophys. 184, 453 (1977). 24 G. I. Rhyu, W. J. Ray, and J. L. Markley, Biochemistry 24, 4746 (1985). 25 j. L. Bock and B. Sheard, Biochem. Biophys. Res. Commun. 66, 24 (1975). 26 W. E. Hull and B. D. Sykes, J. Mol. Biol. 98, 121 (1975). 27 M. Gassner, D. Stehlik, O. Schrecker, W. Hengstenberg, W. Mauer, and H. Ruterjans, Eur. J. Biochem. 75, 287 (1977). 28 G. Dooijewaard, F. Roossien, and G. T. Robillard, Biochemistry 18, 2996 (1979). 29 j. M. Fujitaki, G. Fung, E. Y. Oh, and R. E. Smith, Biochemistry 20, 3658 (1981).
270
PROTEIN STRUCTURE
[13]
dines, as a change in p H is not accompanied by a change in chemical shift. 27 In some highly phosphorylated phosphoproteins, such as phosvitin 3° or the neurofilament proteins, 3~ phosphoserine and phosphothreonine residues can be present simultaneously. These can potentially be differentiated by their chemical shifts but further confirmation can be obtained if the coupling constants can be measured (see below). F o r N M R studies of most nuclei, other than 3~p, there is often a good understanding for the manner in which the binding of certain agents should affect the chemical shift. For example, it would be useful if an upfield shift on the binding of a metal ion in a 31p N M R spectrum could be interpreted as a direct effect of coordination. Unfortunately, however, this cannot be done. The phosphorus chemical shift is very dependent on the bond geometry, on the electronegativity of the substituents, and on the relative amount of 7r bonding. These three parameters are interdependent and their interrelationships are unknown. 32,33 Gorenstein and coworkers 32 have experimentally demonstrated the large effects that bond strain may exert on the chemical shift. Moreover, we have observed that the shifts induced by substituents with varying electronegativity were exactly opposite to what was theoretically predicted based on a simple deshielding model. 33 Thus, although the formation of salt linkages 34 and hydrogen bonds 35 undoubtedly will exert some effects on the chemical shifts, it is presently impossible to predict theoretically in what direction the shift should change. As a result, the interpretation of 31p chemical shifts remains empirical. Coupling Constants Of all the phosphorylated amino acids, only phosphoserine and phosphothreonine display an easily detectable coupling to adjacent protons. In the case of phosphoserine, a three-bond coupling to the t w o / 3 protons results in a triplet with a coupling constant J m c ~ ~ 6.5 Hz. Phosphothreonine has a three-bond coupling to its single P proton and thus displays a d o u b l e t J 6 Broadband or composite pulse proton decoupling 3oH. J. Vogel, Biochemistry 22, 668 (1983). 31U. P. Zimmerman and W. W. Schlaepfer, Biochemistry 25, 3533 (1986). 32D. G. Gorenstein (ed.), "Phosphorus-31 NMR: Principles and Applications," pp. 7-36. Academic Press, Orlando, Florida, 1984. 33H. J. Vogel and W. A. Bridger, Biochemistry 21, 394 (1982). 34K. D. Schnackerz, K. Feldrnann, and W. E. Hull, Biochemistry 18, 1536 (1979). 35F. E. Evans and N. O. Kaplan, FEBS Lett. 105, 11 (1979). 36C. Ho, J. A. Magnuson, J. B. Wilson, N. S. Magnuson, and R. J. Kurland, Biochemistry 8, 2074 (1969).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
271
can be used to remove these coupling effects. The resulting collapse of the signals is often useful, as it improves the signal-to-noise ratio. However, the JPocH coupling-constant information contains valuable information because a Karplus relation exists describing the bond angles for these three-bond couplings. 37,38Thus, a determination of the coupling constant may provide information about preferred side-chain conformations. In the case of ovalbumin a normal bond angle and rotational freedom were observed, 39 but for alkaline phosphatase an unusual rotomer population was involved. 4° For larger proteins with short T2 relaxation times, the coupling is generally not observed. At a higher magnetic field they are also difficult to observe because of the effects of chemical shift anisotropy (see below) on the linewidths. Relaxation Behavior The 3~p nucleus in most biologically relevant molecules is primarily surrounded by 160 and t2C, which would not aid in the relaxation. In phosphoramidates the 14N nucleus to which the 3~p is directly bonded could give rise to some scalar relaxation of the second kind; however, this effect is unlikely to be of great importance for macromolecules with long correlation times. As a result, the two major contributing mechanisms for relaxation in protein-bound phosphorous atoms is through dipole-dipole interactions with neighboring IH, or through chemical shift anisotropy (CSA) mechanisms. 41 In the limit of slow motion (tOpZc > 1), which will apply for most proteins as long as the phosphate group does not rotate freely with respect to the protein surface, the formulas for the linewidth are as follows2,2°,42: Chemical shift anisotropy: Proton-phosphorus dipole-dipole:
Av
=
~
4
2 2 ~2 top(Ao') (1 + -~-)zc (I)
Av = ~1 \ ~ / ~ ' c
t
(2)
37L. D. Hall and R. B. Malcolm, Can. J. Chem. 50, 2102 (1972). 38B. J. Blackburn, R. D. Lapper, and T. C. P. Smith,J. Am. Chem. Soc. 95, 2873 (1973). 39H. J. Vogel and W. A. Bridger,Biochemistry 21, 5825 (1982). 4oj. F. Chlebowski,I. M. Armitage,P. P. Tusa, and J. E. Coleman,J. Biol. Chem. 251, 1207 (1976). 41 In small molecules, contributions of the spin-rotation mechanism to the relaxation cannot be ignored. For a discussion, see, for example, P. Bendel and T. L. James, J. Magn. Reson. 48, 76 (1982) and references therein. 42 B. D. Nageswara Rao, in "Phosphorus-31 NMR: Principles and Applications" (D. G. Gorenstein, ed.), pp. 57-103. Academic Press, Orlando, Florida, 1984.
A
FIG. 1. (A) The N3-phosphohistidine resonance ofEscherichia coli succinate-CoA ligase as recorded at four spectrometers with different field strength. (B) The measured linewidth at half-height plotted against the square of the resonance frequency. The frequency-independent portion of the linewidth is indicated by the dotted line. The contribution of the chemical shift anisotropy is indicated by the solid line and clearly becomes dominant at higher fields. [Reprinted with permission from H. J. Vogel, W. A. Bridger, and B. D. Sykes, z° Biochemistry 21, 1126 (1982). Copyright 1982 American Chemical Society.]
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS B
i
i
273
i
I0O
8o
-.
60
m ii
-1-
40
20
/
H
I
0
40
I
120
I
I
200
280
oj = (MHZ2) FIG. l B .
where Av is the linewidth, ~'c is the correlation time, top is the resonance frequency, Atr(1 + ~qz/3)1/2 is the "anisotropy term," y is the gyromagnetic ratio for different nuclei, and rpH is the distance between phosphorus and proton nuclei. Obviously for the chemical shift anisotropy mechanism the linewidth is linearly dependent on to2. If this relaxation mechanism contributes, the linewidth should increase with increasing resonance frequency. Thus, unlike 13C and ]H, for 31p the highest magnetic field strength does not necessarily give the best resolution and sensitivity. As both dipolar and CSA relaxation mechanisms play a role in relaxation of protein-bound phosphorus nuclei, it will be necessary to determine their relative contributions to the relaxation before ~'c can be obtained from relaxation measurements. This can be accomplished by performing frequency-dependent measurements, where the linewidth is measured at various field strengths (see Fig. 1). Such an analysis has only been performed in a few instances; it has been demonstrated that at lower field (2.3 T) the dipolar mechanism is dominant, whereas at higher field (9.4 T) the linewidth is mainly determined by the chemical shift anisotropy mechanism. 2°,39 Although this situation will not be as severe for relatively mobile phosphate groups, similar problems will be encountered for most phosphoproteins.
274
PROTEIN STRUCTURE
[13]
It should be noted that in contrast to the linewidth (-l/T2), the T1 originating from CSA is not necessarily field dependent, but that the TI for dipolar mechanisms is inversely related to oJ2 .42 Thus an additional measurement of TI may be helpful in diagnosing the contributions from the various relaxation mechanisms. A combination of both T~ and linewidth measurements should give greater confidence to the analysis; if only the latter is used, one has to consider the possibility that an increasing linewidth with to2 could also be caused by chemical exchange, z° Heteronuclear nuclear Overhauser effects (NOEs) from IH to 31p for phosphoproteins have received relatively little attention. 43 Close to the maximum NOE can be observed in highly mobile phosphate groups under conditions where the heteronuclear dipolar relaxation mechanisms are still dominant. However, it should collapse relatively fast when measurements are done at higher magnetic field and motions are slow. 43 pH Titrations The data in Table II show that the majority of all phosphoamino acids display a pH dependence for the chemical shift, the only exception being the two phosphohistidine moieties. 44 Thus pH titration experiments provide a useful means for studying the solvent exposure of these residues. In addition, information about the pKa values for individual groups can be obtained for proteins which give rise to 31p NMR spectra in which the individual resonances can be resolved) 9 As indicated above, in addition to chemical shift the observation of a typical pH titration behavior provides further support for the identification of the chemical nature of a phosphoamino acid. Not only the pKa values (see Table II) but also the changes in chemical shift (A ppm) with titration are diagnostic for the different classes of phosphoamino acids 45 (see Fig. 2). The absence of a discernible pH titration for phosphoamino acids is usually caused by inaccessibility or by the presence of a strong linkage with a positively charged side chain of lysine or arginine, for example. However, the latter situation does not need to abolish the pH titration. Proximal positive charges may in fact lower the p K a , o r they may give rise to an increase in the Hill coefficient, 46 which describes the cooperativity 43 p. A. Hart, in "Phosphorus-31 NMR: Principles and Applications" (D. G. Gorenstein, ed.), pp. 317-348. Academic Press, Orlando, Florida, 1984. Denaturants such as urea and high temperatures may have some effects on the chemical shifts and pK, values determined in 3tp NMR pH titration experiments. These effects are generally quite small (<0.2 ppm or <0.2 pH units). 45 H. J. Vogel and W. A. Bridger, Can. J. Bioehem. Cell Biol. 61, 363 (1983). 46 j. L. Markley, Acc. Chem. Res. 8, 70 (1975).
A -8 -6 -4
Creatine phos~te
EQ .- 2 Q. v
0 LL
{n 2 .J
-~ 4
:E hi
pflosl~te
"r
o
6
L
'
AMP- NH=
10
-8 -6 E
~.-4
~-2 "l-
_J <~ (.)
I
I
2
4
I
I
I
I
6 pH
8 J"
I0
12
~
rginineo.~sphate arbamyl pl~phate •
0
UJ -r
in
4
phosphate
6 I
I
I
4
8
12
pH
FIG. 2. (A and B) 3]p NMR pH titration curves for a series of phosphorus-containing model compounds that resemble phosphoamino acids. (Reproduced with permission from Vogel and Bridger. 45)
276
PROTEIN STRUCTURE
[13]
of the titration. 45 Alternatively, proximal negative charges could result in increases in the determined PKa, or in a decrease in the Hill coefficient.45 For the highly acidic protein phosvitin, for example, we have measured a fairly normal pK~ but a very low Hill coefficient, indicative of the fact that the many negative charges on this protein influence each others' titration behavior. 3° If the neighboring group has a ApKa > 2, a separate inflection in the titration curve may be observed. 47 From a comparison of titration data, it is sometimes even possible to estimate the maximum free energy that is involved in the formation of salt linkages. 45 Results obtained by 31p NMR pH titration experiments may be further supported by FTIR experiments 48,49 in which the protonation state of the phosphate group can be directly followed. Thus, combining the two techniques allows one to distinguish readily between the direct effects of protonation or other conformational effects. A final note of caution should be added: Observation of a change in chemical shift does not directly imply a protonation/deprotonation event on the phosphate group. For example, a phosphate moiety introduced as a modification into the active site of serine proteases displayed a clear pH titration, despite the fact that this compound does not have a pKa within this range. These changes were attributed to the titration of neighboring groups.5°,Sl Thus the possible effects of such interactions or the possibility of pH-induced conformational changes should always be taken into consideration. A final question to be addressed in this section is whether, in the absence of a pH titration for a phosphomonoester moiety, a measurement of the chemical shift alone is sufficient to decide whether a residue is in its protonated or deprotonated form. This has been a contentious issue for some time. Naturally in the case of alkaline phosphatase, where a strained phosphoryl group gives rise to an unusual chemical shift, such information cannot be obtained. 25,26,4° However, in most other proteins and enzymes that have been studied, including all the pyridoxal phosphatecontaining enzymes, the chemical shifts have been fairly close to those observed for model compounds, thus giving support to the notion that information about the chemical shift alone can be taken as support for the 47 R. I. Shrager, J. S. Cohen, R. S. Heller, D. H. Sachs, and A. N. Schechter, Biochemistry 11, 541 (1972). 48 j. M. Sanchez-Ruiz and M. Martinez-Carrion, Biochemistry 25, 2915 (1986). 49 j. M. Sanchez-Ruiz and M. Martinez-Carrion, Biochemistry 27, 3338 (1988). 50 M. A. Porubcan, W. A. Westler, I. B. Ibanez, and J. L. Markley, Biochemistry 18, 4108 (1979). 5~ A. C. M. Van der Drift, H. C. Beck, W. H. Dekker, A. G. Hulst, and E. R. J. Wils, Biochemistry 24, 6894 (1985).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
277
protonation state. Phosphodiester linkages can also readily be distinguished by 31p NMR because they have no pKa above pH 3.0 and as a result they should not titrate. Although the occurrence of such cross-links in proteins is not widespread, they have been found in some prokaryotic proteins.52 31p NMR has been used to identify and study such linkages in the proteins flavodoxin53,54 and glucose oxidase. 55 Effects of Substrates and Other Ligands The binding of substrates to the active site of enzymes that have a stable covalent phosphoamino acid catalytic intermediate may result in changes in the chemical shift and the ]inewidth for the phosphoamino acid. For example, for the Krebs cycle enzyme succinate-CoA ligase, it was observed that the addition of coenzyme A caused a large broadening of the N3-phosphohistidyl resonance. 2~The subsequent addition of a nonmetabolizable analog of the second substrate succinate to form the ternary complex reduced the linewidth again to a value indicative of a tightly held residue, with no mobility with respect to the enzyme (see Fig. 3). These data have been interpreted in terms of a mechanism where the phosphohistidine can adopt two different conformations; exchange between these two is induced by the binding of coenzyme A. One of the conformations, which is dominant only when both coenzyme A and succinate are present, is thought to favor the in-line nucleophilic attack which generates the transient succinyl-P intermediate. 2~The other conformation was thought to facilitate the phosphorylation of the histidine by ATP. Also in the case of phosphoglucomutase, two exchanging conformations have been observed for the active-site phosphoserine. 24 For the amino acids that are phosphorylated by kinases, little effect has been observed when ligands were added. Thus, in the case of the riboflavin-binding protein, the binding of riboflavin did not cause any changes for a series of phosphoserines. 56 However, in the case of xanthine oxidase, a phosphoserine residue was observed which broadened dramatically upon the binding of other ligands. 57 It was therefore suggested that this residue was in the active site of the enzyme and that it was possibly involved as a nucleophile in catalysis. Interestingly, in the case of glycogen phosphorylase, it was observed that addition of the inhibitor 52 S. P. Adler, D. Purich, and E. R. Stadtman, J. Biol. Chem. 250, 6264 (1975). 53 D. E. Edmondson and T. L. James, Proc. Natl. Acad. Sci. U.S.A. 76, 3786 (1979). 54 C. T. Moonen and F. Muller, Biochemistry 21, 408 (1982). 55 T. L. James, D. E. Edmondson, and M. Husain, Biochemistry 20, 617 (1981). 56 M. S. Miller, M. T. Mas, and H. B. White, Biochemistry 23, 569 (1984). 57 M. D. Davis, D. E. Edmondson, and F. Muller, Eur. J. Biochem. 145, 237 (1984).
PROTEIN STRUCTURE
278
[13]
oL_
L
A
' 1 ' 1
. . . .
15
I
10
.
.
.
.
.
11
. . . .
0
I
-5
. . . .
I
- 10
~ ~
t
PPH
FIG. 3.31p NMR spectra of Escherichia coli succinate-CoA ligase (A) in the presence of the ATP analog AMPPCP, (B) plus Mg 2÷, (C) plus coenzyme A, and (D) plus 2,2-difluorosuccinate, a competitive analog of the substrate succinate. The phosphohistidine resonance is around - 5 ppm, shifts upfield on addition of Mg 2÷, broadens and shifts downfield with coenzyme A, and narrows and moves upfield again when the difluorosuccinate is added. The ATP methylene analog was used here because it is nonhydrolyzable and its 3, resonance does not overlap with the His-P resonance, unlike the one for ATP3'. The assignments of the resonances in these spectra are AMPPCP c~ ( - 1 0 ppm), AMPPCP/3 and 3' (10 and 15 ppm, dependent on Mg2+), coenzyme A phosphomonoester (4 ppm), and phosphodiester ( - 1 0 ppm). (From Vogel and Bridger. 2~)
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
279
glucose increased the mobility for the regulatory phosphoserine-14. 58,59 Although glucose binds to the active site, which is far removed from this regulatory site, it was known that the hydrolysis of the phosphoserine by the protein phosphatase was drastically increased by the glucose addition. 6° Thus 31p NMR provided the rationale for this observation: the binding of glucose to the remote active site increases the mobility and surface exposure of the serine phosphate, allowing the phosphatase to act. One of the problems that can arise when substrates and other ligands are added is that these added components can be phosphorylated. Thus one may run into problems because of overlap of resonances for substrates and phosphoamino acids. Fortunately, these problems can be easily solved because a large number of commercially available analogs for compounds such as ATP, ADP, and AMP have substituted phosphorus groups. As these substitutions bring about characteristic changes in the chemical shift (see Table III), 33,59,6|,62 overlap of resonances can be avoided through a judicious choice of analog (see Fig. 3). The use of these analogs has some further advantages. For example, the bridging methylene and imido substitutions provide for nonhydrolyzable ATP and ADP analogs. Not only does this limit the enzymatic turnover, which would normally complicate the lengthy NMR experiments, but if it can be demonstrated that the analog is a competitive inhibitor, one can also differentiate between events caused by the binding of ATP or phosphorylation by this nucleotide. Also, the slow rate at which thio analogs are used as substrates by many enzymes can be used to advantage, because one can potentially follow the slow turnover. A somewhat different ligand than those discussed above is the electron, which can be introduced into the isoalloxazine ring of the FAD and FMN cofactors of redox proteins. The unpaired electron spin may cause broadening effects on the 31p NMR resonances, which can subsequently be used to estimate the average distance between the electron and the phosphate group. 53-55 Binding of Metal Ions The majority of enzymatic phosphoryl-transfer reactions are strictly dependent on the presence of divalent cations such as Mg2+.63 Not only is 58M. Hoerl, K. Feldmann,K. D. Schnackerz, and E. J. M. Helmreich,Biochemistry 18, 2457 (1979). 59S. G. Withers, N. B. Madsen, and B. D. Sykes,Biochemistry 20, 1748(1981). 6oN. B. Madsen, Enzymes 17, 365 (1986). 61E. K. Jaffe and M. Cohn, Biochemistry 17, 652 (1978). 62S. Tran Dinh and M. Roux, Eur. ]. Biochem. 76, 245 (1977). 63A. S. Mildvan,Adv. Enzymol. 49, 103 (1979).
280
PROTEIN STRUCTURE
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TABLE III 3~p NMR PARAMETERS AND pKa VALUES OF SUBSTITUTED PHOSPHORUS COMPOUNDS
Compound
Change in chemical shifts (ppm) a
Change in pK, b
Thiophosphates
-40
Fluorophosphates
+ 11
No c
Imidophosphates
- 10
+0.9
Methylene phosphates
-25
+ 1.5
9
No c
Cyclic phosphates
-
1,5
Analogs available Nonbridging substitution, slowly hydrolyzable Terminal group of ATP, ADP, and AMP Bridging substitution, nonhydrolyzable Bridging substitution, nonhydrolyzable Nonhydrolyzable, shift depends on bond angle
Reference 61
33
62
33
59
a Negative values indicate a downfield shift. b Negative values indicate a decrease in pKa. c No pH titration occurs between pH 3 and 10; both the fluoro and cyclic phosphate analogs are monoanionic and therefore they do not closely resemble the natural compounds, which are generally mainly dianionic at pH 7.0.
the ATP-Mg 2+ complex (rather than ATP) the preferred substrate, but some of the enzymes are metaUoenzymes and contain tightly bound Z n 2+ and Mg 2+ ions. Thus there has been a considerable interest in using NMR techniques to determine the role of the metal ion. As Zn 2+ does not have useful NMR properties, it has been replaced with H3Cd2+, which is a spin½nucleus. 64 For phosphoglucomutase64 and alkaline phosphatase 65-67 (and w i t h llZCd2+ or 114Cd2+ as a no-spin control), 113Cd-3JP couplings have been observed which provide evidence for the direct coordination of the metal ion to the phosphate group. Unfortunately, however, in our hands this approach has not worked well with enzymes wherein Mg 2+ is not tightly bound to the protein. A second substitution that is often used is the paramagnetic Mn 2+ in place of Mg 2+. Introduction of this cation generally 64 G. I. Rhyu, W. J. Ray, and J. L. Markley, Biochemistry 23, 252 (1984). 65 j. D. Otvos, J. R. Alger, J. E. Coleman, and I. M. Armitage, J. Biol. Chem. 254, 1778 (1979). 66 p. Gettins and J. E. Coleman, J. Biol. Chem. 258, 408 (1983). 67 p. Gettins and J. E. Coleman, J. Biol. Chem. 259, 4991 (1984).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
281
produces a broadening that can be used to obtain distance information. Mn 2÷, as well as the stable paramagnetic nitroxides, have also been used as general broadening reagents, to determine whether a residue is on the surface or is deeply buried within the protein. The latter residues in general do not experience any line broadening when such agents are added to the solution. If they are on the surface, broadening is generally observed. ~3,55 Conclusions and Other Experiments In the foregoing discussions we have considered how the nature, mobility, and titratability of phosphoproteins can be determined and how these are affected by ligands and metal ions. However, some other types of experiments have also been performed. In the case of the enzyme alkaline phosphatase, both covalent E - P and noncovalent E- P intermediates can be detected in the 3~p NMR spectra. Because these were well separated, it was possible to perform saturation transfer experiments, which allowed the authors to determine the interconversion rates between these catalytic intermediates. 65 Solid-state NMR experiments have also been performed using phosphoproteins. 68These have given support to the idea that there is little difference between the phosphate groups in the protein in solution or in a dried form. Finally, it should be mentioned that the technique of proton detection of nuclei such as t3C and ~SN could also prove useful for studying 31p.69 Unfortunately, however, only in the case of phosphoserine and phosphothreonine can a direct coupling be observed between the proton and phosphorus nuclei; thus it is likely that this technique will remain limited to the study of these two phosphoamino acids. In cases where information is available about the mobility and titratability of the phosphoamino acids, some interesting biochemical generalizations have come forward. Severe restrictions of motions usually occur in the active sites of enzymes. 2 This is consistent with the notion that the immobility facilitates the in-line nucleophilic attack 1° which is known to occur in all phosphoryl transfer enzymes. The fact that most of these sites do not titrate when ligands are bound suggests that these groups are generally shielded from the solvent. Conversely, flexibility and pH titratability were observed for all the sites that were covalently phosphorylated by protein kinasesf1,7°,7~ This flexibility may play an important role allow68 L. J. Banaszak and J. Seelig, Biochemistry 21, 2436 (1982). 69 A. Bax, this series, Vol. 176 [8]. 7o S. P. Williams, W. A. Bridger, and M. N. G. James, Biochemistry 25, 6655 (1986). 7t M. W. Killimann, K. D. Schnackerz, and M. G. Heilmeyer, Biochemistry 23, 112 (1984).
282
PROTEIN STRUCTURE
[14]
ing access of protein kinases and protein phosphatases to the regulatory site. All regulatory phosphorylation sites are dianionic at physiological pH. If salt linkages with basic amino acid side chains occur, the free energy involved TM is AG ° ~< - 5 . 0 kcal mo1-1. However, it is also possible that the highly charged and mobile phosphoryl group would prevent the interaction between two hydrophobic domains on a protein and exert its regulatory function in this fashion. Acknowledgments Research on regulatory proteins in the author's laboratory is presently sponsored by a grant from the Medical Research Council of Canada. The author is the recipient of a Scholarship from the Alberta Heritage Foundation for Medical Research. The secretarial assistance of Susan Clegg is greatly appreciated.
[14] I s o t o p i c L a b e l i n g w i t h H y d r o g e n - 2 a n d C a r b o n - 1 3 to Compare Conformations of Proteins and Mutants G e n e r a t e d b y S i t e - D i r e c t e d M u t a g e n e s i s , II
By JOYCE A. WILDE, PHILIP H. BOLTON, DAVID W. HIBLER, L Y N N HARPOLD, TAYEBEH POURMOTABBED, M A R K D E L L ' A C Q U A , a n d JOHN A . GERLT
Introduction Our preceding chapter in this volume [4] describes the preparation of isotopically labeled staphylococcal nuclease (Snase) samples as well as the characterization of these samples by one-dimensional NMR methods. The one-dimensional NMR spectra clearly show that Snase samples of high isotopic enrichment can be prepared in amounts suitable for NMR studies. In this chapter we describe the strategy for using isotopic labeling to determine the effects of site-specific amino acid replacements on the conformation of wild-type and mutant proteins. A focus of our research has been the investigation of the effects of sitespecific mutations on the activity and conformation of Snase. 1 At the beginning of this project we were confident that active-site mutants of Snase could be generated and that their enzymatic activity could be as1 D. W. Hibler, N. J. Stolowich, M. A. Reynolds, J. A. Gerlt, J. A. Wilde, and P. H. Bolton, Biochemistry 26, 6278 (1987).
METHODS IN ENZYMOLOGY, VOL. 177
Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
263
information on the distance of a nucleus from the metal, and NOE contains information on the distance between two nuclei, this approach may allow us to redesign the environment of metal ions in proteins, i.e., to design the active cavity of metalloenzymes. This information, coupled with molecular dynamics, is an independent, indispensable approach to the investigation of protein structures. Such studies can also be performed on t3C, J9F, and 3~p nuclei of the protein or of a substrate interacting with a protein. The window of paramagnetic compounds that can be investigated is enlarged by magnetically coupled systems, either natural or artificial. A wealth of information may be obtained on the coupling and on the effects of the coupling on electron relaxation.
[13] P h o s p h o r u s - 3 1 N u c l e a r M a g n e t i c Resonance of Phosphoproteins By HANS J. VOGEL
Introduction The ribosomal protein synthesis machinery utilizes the standard 20 amino acids as the building blocks for proteins. The five elements that are present in these amino acids are hydrogen, carbon, nitrogen, oxygen, and sulfur. Consequently, all proteins can in principle be studied by ill, 13C, |4N/15N, 170, or 33S NMR techniques. Few studies of proteins, however, have utilized 14N, 170, or 33S NMR; the electric quadrupole moment of these nuclei renders resonances for the individual atoms very broad, leading to featureless unresolved spectra.~ Because of their more favorable nuclear properties (spin -- ½), the study of the other three nuclei has provided considerable insight into structural, functional, and dynamic properties of proteins, as will be apparent from the various chapters in this volume. As the element phosphorus is not incorporated during protein biosynthesis, it can only become incorporated into proteins through various posttranslational processes. Consequently only a few phosphorus atoms are found in most phosphoproteins. This has the advantage that the problem of resonance assignment, which can be quite formidable for ~H, ~aC, and ~SN NMR studies of larger proteins, normally does not pose any S. Forsen and B. Lindman, Methods Biochem. Anal. 27, 289 (1981). METHODS IN ENZYMOLOGY, VOL. 177
Copyright © 1989 by Academic Press, lnc. All rights of reproduction in any form reserved.
264
PROTEIN STRUCTURE
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TABLE I PROPERTIES OF SOME COMMON NUCLEI
Nucleus
Resonance frequency (MHz) °
Natural abundance (%)
Sensitivity b
Chemical shift range for biomolecules (ppm)
1H 13C 15N 31p
400.1 100.6 40.5 162.0
99.98 1.11 0.37 100.0
1.00 0.016 0.001 0.066
15 200 100 20
At 9.4 T. b This is for an equal number of nuclei. The receptivity that actually governs the sensitivity of the NMR experiment (and which is usually defined as the isotopic abundance multiplied by the sensitivity) is less for nuclei with low isotopic abundance. a
serious problems with 31p NMR.2 Furthermore, because of the paucity of resonances in 31p N M R spectra of phosphoproteins, it has been possible to pursue studies of proteins with molecular weights up to 500,000. 2 In contrast, the problem of resonance overlap, which is inherent in IH, ~3C, and 15N N M R studies of larger proteins, severely limits the size of protein that can be studied by these three techniques. On the other hand, the information that can be extracted by 3tp NMR is of a different nature than the overall structural detail that can potentially be obtained by the other techniques. In addition to the different way in which phosphorus is incorporated into proteins, it is important to realize that the s~P nucleus has a favorably high resonance frequency and a good sensitivity for NMR experiments (see Table I). It is also 100% naturally abundant, and thus can be studied without the need for introducing isotopic labels. Isotopic labeling can be expensive and experimentally cumbersome, and these factors have limited the use of nuclei with low natural abundance, as in 13C or ~SN NMR. 3 The combination of the biological and physical factors mentioned above explains why 3~p NMR has become such a useful tool for the study of phosphoproteins. In this chapter we will focus on the practical aspects of the technique, which will be illustrated by some selected examples. It is 2 H. J. Vogel, in "Phosphorus-31 NMR: Principles and Applications" (D. G. Gorenstein, ed.), pp. 104-154. Academic Press, Orlando, Florida, 1984. 3 Modern NMR pulse methods can be used to study indirectly tSN or t3C nuclei, by utilizing heteronuclear quantum coherences and detecting the attached protons. These methods provide a great increase in sensitivity, which should overcome to some extent the need for incorporating isotopic labels.
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
265
not our intent to give an exhaustive review, and the reader wishing to obtain more details should consult other sources 2,4 or the original literature. Before discussing the experimental aspects of 31p NMR of phosphoproteins, we will briefly review the biological mechanisms through which phosphate can be incorporated into proteins. Phosphorus can become part of a protein through essentially three different mechanisms. The first mechanism is the incorporation of phosphorylated coenzyme into a protein. Such moieties are either extremely rigidly bound to the protein or in some cases they become covalently attached to the side chain of specific amino acids. An obvious example is the coenzyme pyridoxal phosphate, which occurs in the active site of a large number of enzymes. 5 Although the phosphate group of this coenzyme does not appear to be directly involved in enzymatic catalysis, the dephospho (pyridoxal) form generally does not support catalysis. 6,7 As such, the phosphate group presents a nonperturbing spectral reporter group for studying events that take place in the active site of the enzyme. Also, the cofactors FMN and FAD, which are part of many redox proteins, carry phosphate groups that can be studied by 31p NMR. The other two groups of phosphoproteins contain phosphorylated amino acids rather than coenzymes. Of particular interest is a large group of proteins and enzymes whose activity is regulated by reversible enzymatic phosphorylation/dephosphorylation reactions. Specialized protein kinases that can be activated by hormonal stimulation can transfer a phosphoryl group (PO32-) from ATP onto unique serine, threonine, or tyrosine residues on selected target proteins. 8,9 In some nuclear proteins unique histidine, lysine, and arginine side chains can also become phosphorylated. Although these regulatory phosphorylation sites are often far removed from the active site of these enzymes, the phosphorylation has marked effects on the activity. The phosphoryl groups can subsequently be removed by specialized protein phosphatases that are also hormonally controlled. In addition to these regulatory phosphorylations there is a series of food storage proteins in milk and eggs (ovalbumin, casein, and phosvitin) and a series of polyelectrolyte proteins (bone, tooth, and saliva phosphoproteins) that are also phosphorylated by protein kinases. How4 T. L. James, CRC Crit. Rev. Biochem. 18, 1 (1985). 5 K. D. Schnackerz and E. E. Snell, J. Biol. Chem. 258, 4839 (1983). 6 M. E. Mattingly, J. R. Mattingly, and M. Martinez-Carrion, J. Biol. Chem. 257, 8872 (1982). 7 S. G. Withers, N. B. Madsen, B. D. Sykes, M. Takagi, S. Shimomura, and T, Fukui, J. Biol. Chem. 256, 10759 (1981). 8 p. Cohen, Eur. J. Biochem. 151, 439 (1985). 9 A. M. Edelman, D, K. Blumenthal, and E. G. Krebs, Annu. Rev. Biochem. 56, 567 (1987).
266
PROTEIN STRUCTURE
[13]
ever, in this case, the phosphorylation does not play a regulatory role and the proteinous phosphate group is either involved in the binding and precipitation of metal ions or simply functions as a phosphate store. The second group of phosphoproteins, which contain phosphoamino acids, consists of enzymes in which the phosphorylated amino acid is an enzymatic catalytic intermediate.i° The phosphoryl group is transferred from substrates (such as ATP, for example) onto an amino acid side chain in the active site. The residues that become phosphorylated are either a serine (as in the enzymes alkaline phosphatase and phosphoglucomutase), a histidine (as in HPr, succinate-CoA ligase, phosphoglycerate mutase, ATP citrate-lyase), or an aspartate (which has been observed for the Ca 2+-, Na÷,K +-, and the H÷,K+-ATPase). Because they are intermediates in catalysis, their formation is of a transient nature during active turnover. Nevertheless, many of these enzymes can be purified in a stable phosphorylated form that can be studied by 31p NMR. In cases where the covalent phosphoamino acid is unstable, other techniques involving the use of the oxygen isotopes 180 and 170 may be useful to determine whether a phosphorylated intermediate exists 1°,11 and whether it is an obligatory intermediate in catalysis.12 In the following discussion we examine the NMR parameters that can be measured by 31p NMR. We will mainly focus on proteins that contain phosphoamino acids, but some examples involving bound coenzymes will also be mentioned. Sample Preparations For most proteins it will be necessary to make up a 1.5-ml sample with a protein concentration of ->0.2 mM. This sample size is necessary to fill a 10-mm NMR tube to a level that adequately covers the receiver coil and to obtain a signal in a reasonable time (several hours). For proteins that give rise to relatively narrow resonances, it is possible to use protein concentrations that are somewhat lower. The relatively high protein concentration may sometimes result in protein aggregation. In such cases it is advisable to use a larger NMR tube and to reduce the protein concentration while keeping the same amount of protein within the area covered by the receiver coil. For all samples, it is usually advisable to use a Teflon plug, to avoid a vortex in the sample while spinning. Because most phosphorylated compounds carry a negative charge, precautions have to be 10j. R. Knowles,Annu. Rev. Biochem. 49, 877 (1980). ~lj. j. Villafranca, this volume [20]. 12M. J. Wimmerand I. A. Rose, Annu. Rev. Biochem. 47, 1031 (1978).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
267
taken to exclude positively charged paramagnetic metal ions such as Mn 2÷ and Ni 2÷. In order to avoid any such problems, it is therefore advisable to treat all the buffers--and even the 10-20% D20 which is normally used for lock purposes--with a chelating agent such as Chelex 100. It is also of extreme importance to ascertain that the sample does not contain any phosphorylated impurities. For example, nucleic acids or lipids can sometimes be difficult to extract and they can copurify with a protein. The phosphodiester linkages in these molecules give rise to peaks in the 31p NMR spectrum. Extreme conditions, such as organic solvents, are sometimes necessary to remove such impurities. In one case we even were forced to precipitate the protein to prove that a phosphodiester linkage was not part of a protein, but rather a lipid impurity. 13 Not only nucleic acids and lipids can cause problems. For example, the extremely tight binding of the nucleotides AMP or IMP to glycogen phosphorylase necessitates a dialysis procedure against activated charcoal to remove these contaminants. 7 If such precautions are not taken, erroneous results can occur. For example, Fossel et al.14 reported a highly unusual chemical shift for the catalytic phosphoaspartate in the active site of a Na ÷, K ÷ATPase. The enthusiasm for this intriguing observation was severely tempered when subsequent studies showed that the resonance was in fact an ATP contamination and that the real intermediate had a very normal chemical shift. 15Finally, it is important to ascertain that the sample is free from protease and phosphatase activity. As it may be in the spectrometer for several days at temperatures above 4°, it should preferably also be sterile to prevent growth of microorganisms. In some cases addition of a small amount of an antibiotic may be useful. If these conditions are not met, degradation reactions may take place in the course of the experiment, which will generally affect the experimental outcome. Some covalent enzymatic phosphoamino acid intermediates may be intrinsically unstable; this will lead to the gradual disappearance of their resonance and to the appearance of a Pi peak in the spectrum. With respect to the pulsing conditions under which spectra should be collected, it is difficult to generalize. In our experience T~ values for phosphoproteins can vary between 0.25 and 4 sec. Thus the optimal NMR parameters will need to be determined for each individual case. 13 G. D. Armstrong, L. S. Frost, H. J. Vogel, and W. Paranchych, J. Bacteriol. 145, 1167 (1981). 14 E. T. Fossel, R. L. Post, D. S. O'Hara, and T. W. Smith, Biochemistry 20, 7215 (1981). 15 G. M. Sontheimer, H. R. Kalbitzer, and W. Hasselbach, Biochemistry 26, 2701 (1987).
268
PROTEIN STRUCTURE
[13]
TABLE II ACID-BASE STABILITY, PKa VALUES, AND 31p N M R PARAMETERS FOR PHOSPHOAMINO ACIDS a
Phosphoamino acid Phosphomonoesters b Phosphoserine Phosphothreonine Phosphotyrosine Phosphodiesters c RNA, DNA, and phospholipids Acyl phosphates Phosphoaspartate Phosphoramidates NS-Phosphohistidine N1-Phosphohistidine Phospholysine Phosphoarginine
Stable to strong acid
Stable to strong base
+ + +
Chemical shift (ppm) Deprotonated
Protonated
pKa
0.6 0.0 -3.3
5.8 5.9 5.8
0 to - 1.5
No
No
-1.5
-6.5
4.8
-4.5 -5.5 ND -3.0
No No ND -5.4
No No ND 4.3
4.6 4.0 1.0
+
+ + + +
All chemical shifts are reported with reference to 85% H3PO4. Upfield shifts are given a negative sign. ND, Not determined. b The chemical shifts and pH titration behavior of the coenzymes pyridoxal and pyridoxamine phosphate are very similar to those of phosphothreonine, whereas for FMN, these parameters resemble those of phosphoserine. c The diphosphodiester FAD (-10.8/-11.3 ppm) also does not show a titration behavior between pH 3 and 10.
C h e m i c a l Shift a n d N a t u r e of P h o s p h o r y l a t e d A m i n o Acids T h e n a t u r e o f a p h o s p h o a m i n o acid in a n e w l y i s o l a t e d p h o s p h o p r o t e i n is g e n e r a l l y d e t e r m i n e d f r o m a s t u d y of its acid a n d b a s e stability (see T a b l e II). 16-18T h i s s i m p l e test n o r m a l l y p r o v i d e s a r e a s o n a b l e first i n d i c a t i o n b u t s o m e c a u t i o n has to b e e x e r c i s e d , b e c a u s e the r e s u l t is k n o w n to b e d e p e n d e n t o n a d j a c e n t a m i n o acids; m o r e o v e r , e l i m i n a t i o n r e a c t i o n s m a y o c c u r . ~6-1s T h e r e f o r e the n e x t step is u s u a l l y the p u r i f i c a t i o n o f a p h o s p h o p e p t i d e , f r o m w h i c h s u b s e q u e n t l y a p h o s p h o a m i n o acid is obt a i n e d a n d identified. 31p N M R p r o v i d e s a n a l t e r n a t i v e n o n i n v a s i v e m e t h o d for d e t e r m i n i n g the n a t u r e o f p h o s p h o a m i n o acids. 2 T h e c h e m i c a l shifts 19 m e a s u r e d for the v a r i o u s a m i n o acids are quite different (see T a b l e 16G. Taborsky, Adv. Protein Chem. 28, 1 (1974). 17T. M. Mortensen, this series, Vol. 107, p. 1. is j. M. Fujitaki and R. A. Smith, this series, Vol. 107, p. 23. 19Chemical shifts in NMR experiments are always measured with respect to a standard. The commonly used standard for 3~p NMR is 85% H3PO4. This standard is not convenient for
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
269
II) and thus they can be used directly for identification purposes. Naturally, chemical shifts are only a direct monitor of the chemical nature of a phosphoamino acid as long as the incorporation into the protein does not perturb the phosphate moiety. By and large no such deviations have been observed for all the regulatory sites where the phosphoryl group has been introduced by the action of a protein kinase. 2 With the exception of alkaline phosphatase, the majority of the covalent enzymatic intermediates have chemical shifts that are relatively close (<2.0 ppm) to those observed for the free amino acid in solution. Thus the chemical shifts for the histidyl-P of succinate-CoA ligase 2°,2z and ATP citrate-lyase, 22 the aspartyl-P of the ATPases, 15and the serine-P of phosphoglucomutase 23,24were all close to their respective standards. Nevertheless, bond strain has been invoked to explain the 3.8-ppm downfield shift for the serine phosphate residue in the active site of alkaline phosphatase 25,z6 and for the 1.5-ppm downfield shift of the active site Nl-phosphate of histidine in the HPr protein. 27,28Thus it is often advisable to determine the chemical shift for the protein in both the native and a denatured state to be absolutely certain. Denaturation can be brought about by urea, pH extremes, etc. Particularly if one wants to differentiate between the N ~- and N3-phos phohistidine, which have quite similar chemical shifts, this is a necessary s t e p . 27,29 Further confirmation for the nature of the phosphoamino acid may be obtained by looking for a characteristic pH dependence of the shift (see below). Although this step is useful for the identification of phosphomonoesters, unfortunately it does not provide help in identifying phosphohisti-
two reasons. First, it falls right in the middle of the spectrum. Second, and most important, the nature of the standard precludes its use as an internal standard. Although capillaries can be used, the high concentration of the standard also makes this impractical for samples that have weak signals. Therefore, we generally include compounds that have a well-characterized shift, such as phosphocreatine or methylene diphosphonate, as internal standards. The latter is conveniently downfield ( - 2 0 ppm) from the biologically occurring compounds, hut the pH dependence of its chemical shift may cause some problems. 20 H. J. Vogel, W. A. Bridger, and B. D. Sykes, Biochemistry 21, 1126 (1982). 21 H. J. Vogel and W. A. Bridger, J. Biol. Chem. 257, 4834 (1982). 22 S. P. Williams, B. D. Sykes, and W. A. Bridget, Biochemistry 24, 5527 (1985). 23 W. J. Ray, A. S. Mildvan, and J. B. Grutzner, Arch. Biochem. Biophys. 184, 453 (1977). 24 G. I. Rhyu, W. J. Ray, and J. L. Markley, Biochemistry 24, 4746 (1985). 25 j. L. Bock and B. Sheard, Biochem. Biophys. Res. Commun. 66, 24 (1975). 26 W. E. Hull and B. D. Sykes, J. Mol. Biol. 98, 121 (1975). 27 M. Gassner, D. Stehlik, O. Schrecker, W. Hengstenberg, W. Mauer, and H. Ruterjans, Eur. J. Biochem. 75, 287 (1977). 28 G. Dooijewaard, F. Roossien, and G. T. Robillard, Biochemistry 18, 2996 (1979). 29 j. M. Fujitaki, G. Fung, E. Y. Oh, and R. E. Smith, Biochemistry 20, 3658 (1981).
270
PROTEIN STRUCTURE
[13]
dines, as a change in p H is not accompanied by a change in chemical shift. 27 In some highly phosphorylated phosphoproteins, such as phosvitin 3° or the neurofilament proteins, 3~ phosphoserine and phosphothreonine residues can be present simultaneously. These can potentially be differentiated by their chemical shifts but further confirmation can be obtained if the coupling constants can be measured (see below). F o r N M R studies of most nuclei, other than 3~p, there is often a good understanding for the manner in which the binding of certain agents should affect the chemical shift. For example, it would be useful if an upfield shift on the binding of a metal ion in a 31p N M R spectrum could be interpreted as a direct effect of coordination. Unfortunately, however, this cannot be done. The phosphorus chemical shift is very dependent on the bond geometry, on the electronegativity of the substituents, and on the relative amount of 7r bonding. These three parameters are interdependent and their interrelationships are unknown. 32,33 Gorenstein and coworkers 32 have experimentally demonstrated the large effects that bond strain may exert on the chemical shift. Moreover, we have observed that the shifts induced by substituents with varying electronegativity were exactly opposite to what was theoretically predicted based on a simple deshielding model. 33 Thus, although the formation of salt linkages 34 and hydrogen bonds 35 undoubtedly will exert some effects on the chemical shifts, it is presently impossible to predict theoretically in what direction the shift should change. As a result, the interpretation of 31p chemical shifts remains empirical. Coupling Constants Of all the phosphorylated amino acids, only phosphoserine and phosphothreonine display an easily detectable coupling to adjacent protons. In the case of phosphoserine, a three-bond coupling to the t w o / 3 protons results in a triplet with a coupling constant J m c ~ ~ 6.5 Hz. Phosphothreonine has a three-bond coupling to its single P proton and thus displays a d o u b l e t J 6 Broadband or composite pulse proton decoupling 3oH. J. Vogel, Biochemistry 22, 668 (1983). 31U. P. Zimmerman and W. W. Schlaepfer, Biochemistry 25, 3533 (1986). 32D. G. Gorenstein (ed.), "Phosphorus-31 NMR: Principles and Applications," pp. 7-36. Academic Press, Orlando, Florida, 1984. 33H. J. Vogel and W. A. Bridger, Biochemistry 21, 394 (1982). 34K. D. Schnackerz, K. Feldrnann, and W. E. Hull, Biochemistry 18, 1536 (1979). 35F. E. Evans and N. O. Kaplan, FEBS Lett. 105, 11 (1979). 36C. Ho, J. A. Magnuson, J. B. Wilson, N. S. Magnuson, and R. J. Kurland, Biochemistry 8, 2074 (1969).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
271
can be used to remove these coupling effects. The resulting collapse of the signals is often useful, as it improves the signal-to-noise ratio. However, the JPocH coupling-constant information contains valuable information because a Karplus relation exists describing the bond angles for these three-bond couplings. 37,38Thus, a determination of the coupling constant may provide information about preferred side-chain conformations. In the case of ovalbumin a normal bond angle and rotational freedom were observed, 39 but for alkaline phosphatase an unusual rotomer population was involved. 4° For larger proteins with short T2 relaxation times, the coupling is generally not observed. At a higher magnetic field they are also difficult to observe because of the effects of chemical shift anisotropy (see below) on the linewidths. Relaxation Behavior The 3~p nucleus in most biologically relevant molecules is primarily surrounded by 160 and t2C, which would not aid in the relaxation. In phosphoramidates the 14N nucleus to which the 3~p is directly bonded could give rise to some scalar relaxation of the second kind; however, this effect is unlikely to be of great importance for macromolecules with long correlation times. As a result, the two major contributing mechanisms for relaxation in protein-bound phosphorous atoms is through dipole-dipole interactions with neighboring IH, or through chemical shift anisotropy (CSA) mechanisms. 41 In the limit of slow motion (tOpZc > 1), which will apply for most proteins as long as the phosphate group does not rotate freely with respect to the protein surface, the formulas for the linewidth are as follows2,2°,42: Chemical shift anisotropy: Proton-phosphorus dipole-dipole:
Av
=
~
4
2 2 ~2 top(Ao') (1 + -~-)zc (I)
Av = ~1 \ ~ / ~ ' c
t
(2)
37L. D. Hall and R. B. Malcolm, Can. J. Chem. 50, 2102 (1972). 38B. J. Blackburn, R. D. Lapper, and T. C. P. Smith,J. Am. Chem. Soc. 95, 2873 (1973). 39H. J. Vogel and W. A. Bridger,Biochemistry 21, 5825 (1982). 4oj. F. Chlebowski,I. M. Armitage,P. P. Tusa, and J. E. Coleman,J. Biol. Chem. 251, 1207 (1976). 41 In small molecules, contributions of the spin-rotation mechanism to the relaxation cannot be ignored. For a discussion, see, for example, P. Bendel and T. L. James, J. Magn. Reson. 48, 76 (1982) and references therein. 42 B. D. Nageswara Rao, in "Phosphorus-31 NMR: Principles and Applications" (D. G. Gorenstein, ed.), pp. 57-103. Academic Press, Orlando, Florida, 1984.
A
FIG. 1. (A) The N3-phosphohistidine resonance ofEscherichia coli succinate-CoA ligase as recorded at four spectrometers with different field strength. (B) The measured linewidth at half-height plotted against the square of the resonance frequency. The frequency-independent portion of the linewidth is indicated by the dotted line. The contribution of the chemical shift anisotropy is indicated by the solid line and clearly becomes dominant at higher fields. [Reprinted with permission from H. J. Vogel, W. A. Bridger, and B. D. Sykes, z° Biochemistry 21, 1126 (1982). Copyright 1982 American Chemical Society.]
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS B
i
i
273
i
I0O
8o
-.
60
m ii
-1-
40
20
/
H
I
0
40
I
120
I
I
200
280
oj = (MHZ2) FIG. l B .
where Av is the linewidth, ~'c is the correlation time, top is the resonance frequency, Atr(1 + ~qz/3)1/2 is the "anisotropy term," y is the gyromagnetic ratio for different nuclei, and rpH is the distance between phosphorus and proton nuclei. Obviously for the chemical shift anisotropy mechanism the linewidth is linearly dependent on to2. If this relaxation mechanism contributes, the linewidth should increase with increasing resonance frequency. Thus, unlike 13C and ]H, for 31p the highest magnetic field strength does not necessarily give the best resolution and sensitivity. As both dipolar and CSA relaxation mechanisms play a role in relaxation of protein-bound phosphorus nuclei, it will be necessary to determine their relative contributions to the relaxation before ~'c can be obtained from relaxation measurements. This can be accomplished by performing frequency-dependent measurements, where the linewidth is measured at various field strengths (see Fig. 1). Such an analysis has only been performed in a few instances; it has been demonstrated that at lower field (2.3 T) the dipolar mechanism is dominant, whereas at higher field (9.4 T) the linewidth is mainly determined by the chemical shift anisotropy mechanism. 2°,39 Although this situation will not be as severe for relatively mobile phosphate groups, similar problems will be encountered for most phosphoproteins.
274
PROTEIN STRUCTURE
[13]
It should be noted that in contrast to the linewidth (-l/T2), the T1 originating from CSA is not necessarily field dependent, but that the TI for dipolar mechanisms is inversely related to oJ2 .42 Thus an additional measurement of TI may be helpful in diagnosing the contributions from the various relaxation mechanisms. A combination of both T~ and linewidth measurements should give greater confidence to the analysis; if only the latter is used, one has to consider the possibility that an increasing linewidth with to2 could also be caused by chemical exchange, z° Heteronuclear nuclear Overhauser effects (NOEs) from IH to 31p for phosphoproteins have received relatively little attention. 43 Close to the maximum NOE can be observed in highly mobile phosphate groups under conditions where the heteronuclear dipolar relaxation mechanisms are still dominant. However, it should collapse relatively fast when measurements are done at higher magnetic field and motions are slow. 43 pH Titrations The data in Table II show that the majority of all phosphoamino acids display a pH dependence for the chemical shift, the only exception being the two phosphohistidine moieties. 44 Thus pH titration experiments provide a useful means for studying the solvent exposure of these residues. In addition, information about the pKa values for individual groups can be obtained for proteins which give rise to 31p NMR spectra in which the individual resonances can be resolved) 9 As indicated above, in addition to chemical shift the observation of a typical pH titration behavior provides further support for the identification of the chemical nature of a phosphoamino acid. Not only the pKa values (see Table II) but also the changes in chemical shift (A ppm) with titration are diagnostic for the different classes of phosphoamino acids 45 (see Fig. 2). The absence of a discernible pH titration for phosphoamino acids is usually caused by inaccessibility or by the presence of a strong linkage with a positively charged side chain of lysine or arginine, for example. However, the latter situation does not need to abolish the pH titration. Proximal positive charges may in fact lower the p K a , o r they may give rise to an increase in the Hill coefficient, 46 which describes the cooperativity 43 p. A. Hart, in "Phosphorus-31 NMR: Principles and Applications" (D. G. Gorenstein, ed.), pp. 317-348. Academic Press, Orlando, Florida, 1984. Denaturants such as urea and high temperatures may have some effects on the chemical shifts and pK, values determined in 3tp NMR pH titration experiments. These effects are generally quite small (<0.2 ppm or <0.2 pH units). 45 H. J. Vogel and W. A. Bridger, Can. J. Bioehem. Cell Biol. 61, 363 (1983). 46 j. L. Markley, Acc. Chem. Res. 8, 70 (1975).
A -8 -6 -4
Creatine phos~te
EQ .- 2 Q. v
0 LL
{n 2 .J
-~ 4
:E hi
pflosl~te
"r
o
6
L
'
AMP- NH=
10
-8 -6 E
~.-4
~-2 "l-
_J <~ (.)
I
I
2
4
I
I
I
I
6 pH
8 J"
I0
12
~
rginineo.~sphate arbamyl pl~phate •
0
UJ -r
in
4
phosphate
6 I
I
I
4
8
12
pH
FIG. 2. (A and B) 3]p NMR pH titration curves for a series of phosphorus-containing model compounds that resemble phosphoamino acids. (Reproduced with permission from Vogel and Bridger. 45)
276
PROTEIN STRUCTURE
[13]
of the titration. 45 Alternatively, proximal negative charges could result in increases in the determined PKa, or in a decrease in the Hill coefficient.45 For the highly acidic protein phosvitin, for example, we have measured a fairly normal pK~ but a very low Hill coefficient, indicative of the fact that the many negative charges on this protein influence each others' titration behavior. 3° If the neighboring group has a ApKa > 2, a separate inflection in the titration curve may be observed. 47 From a comparison of titration data, it is sometimes even possible to estimate the maximum free energy that is involved in the formation of salt linkages. 45 Results obtained by 31p NMR pH titration experiments may be further supported by FTIR experiments 48,49 in which the protonation state of the phosphate group can be directly followed. Thus, combining the two techniques allows one to distinguish readily between the direct effects of protonation or other conformational effects. A final note of caution should be added: Observation of a change in chemical shift does not directly imply a protonation/deprotonation event on the phosphate group. For example, a phosphate moiety introduced as a modification into the active site of serine proteases displayed a clear pH titration, despite the fact that this compound does not have a pKa within this range. These changes were attributed to the titration of neighboring groups.5°,Sl Thus the possible effects of such interactions or the possibility of pH-induced conformational changes should always be taken into consideration. A final question to be addressed in this section is whether, in the absence of a pH titration for a phosphomonoester moiety, a measurement of the chemical shift alone is sufficient to decide whether a residue is in its protonated or deprotonated form. This has been a contentious issue for some time. Naturally in the case of alkaline phosphatase, where a strained phosphoryl group gives rise to an unusual chemical shift, such information cannot be obtained. 25,26,4° However, in most other proteins and enzymes that have been studied, including all the pyridoxal phosphatecontaining enzymes, the chemical shifts have been fairly close to those observed for model compounds, thus giving support to the notion that information about the chemical shift alone can be taken as support for the 47 R. I. Shrager, J. S. Cohen, R. S. Heller, D. H. Sachs, and A. N. Schechter, Biochemistry 11, 541 (1972). 48 j. M. Sanchez-Ruiz and M. Martinez-Carrion, Biochemistry 25, 2915 (1986). 49 j. M. Sanchez-Ruiz and M. Martinez-Carrion, Biochemistry 27, 3338 (1988). 50 M. A. Porubcan, W. A. Westler, I. B. Ibanez, and J. L. Markley, Biochemistry 18, 4108 (1979). 5~ A. C. M. Van der Drift, H. C. Beck, W. H. Dekker, A. G. Hulst, and E. R. J. Wils, Biochemistry 24, 6894 (1985).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
277
protonation state. Phosphodiester linkages can also readily be distinguished by 31p NMR because they have no pKa above pH 3.0 and as a result they should not titrate. Although the occurrence of such cross-links in proteins is not widespread, they have been found in some prokaryotic proteins.52 31p NMR has been used to identify and study such linkages in the proteins flavodoxin53,54 and glucose oxidase. 55 Effects of Substrates and Other Ligands The binding of substrates to the active site of enzymes that have a stable covalent phosphoamino acid catalytic intermediate may result in changes in the chemical shift and the ]inewidth for the phosphoamino acid. For example, for the Krebs cycle enzyme succinate-CoA ligase, it was observed that the addition of coenzyme A caused a large broadening of the N3-phosphohistidyl resonance. 2~The subsequent addition of a nonmetabolizable analog of the second substrate succinate to form the ternary complex reduced the linewidth again to a value indicative of a tightly held residue, with no mobility with respect to the enzyme (see Fig. 3). These data have been interpreted in terms of a mechanism where the phosphohistidine can adopt two different conformations; exchange between these two is induced by the binding of coenzyme A. One of the conformations, which is dominant only when both coenzyme A and succinate are present, is thought to favor the in-line nucleophilic attack which generates the transient succinyl-P intermediate. 2~The other conformation was thought to facilitate the phosphorylation of the histidine by ATP. Also in the case of phosphoglucomutase, two exchanging conformations have been observed for the active-site phosphoserine. 24 For the amino acids that are phosphorylated by kinases, little effect has been observed when ligands were added. Thus, in the case of the riboflavin-binding protein, the binding of riboflavin did not cause any changes for a series of phosphoserines. 56 However, in the case of xanthine oxidase, a phosphoserine residue was observed which broadened dramatically upon the binding of other ligands. 57 It was therefore suggested that this residue was in the active site of the enzyme and that it was possibly involved as a nucleophile in catalysis. Interestingly, in the case of glycogen phosphorylase, it was observed that addition of the inhibitor 52 S. P. Adler, D. Purich, and E. R. Stadtman, J. Biol. Chem. 250, 6264 (1975). 53 D. E. Edmondson and T. L. James, Proc. Natl. Acad. Sci. U.S.A. 76, 3786 (1979). 54 C. T. Moonen and F. Muller, Biochemistry 21, 408 (1982). 55 T. L. James, D. E. Edmondson, and M. Husain, Biochemistry 20, 617 (1981). 56 M. S. Miller, M. T. Mas, and H. B. White, Biochemistry 23, 569 (1984). 57 M. D. Davis, D. E. Edmondson, and F. Muller, Eur. J. Biochem. 145, 237 (1984).
PROTEIN STRUCTURE
278
[13]
oL_
L
A
' 1 ' 1
. . . .
15
I
10
.
.
.
.
.
11
. . . .
0
I
-5
. . . .
I
- 10
~ ~
t
PPH
FIG. 3.31p NMR spectra of Escherichia coli succinate-CoA ligase (A) in the presence of the ATP analog AMPPCP, (B) plus Mg 2÷, (C) plus coenzyme A, and (D) plus 2,2-difluorosuccinate, a competitive analog of the substrate succinate. The phosphohistidine resonance is around - 5 ppm, shifts upfield on addition of Mg 2÷, broadens and shifts downfield with coenzyme A, and narrows and moves upfield again when the difluorosuccinate is added. The ATP methylene analog was used here because it is nonhydrolyzable and its 3, resonance does not overlap with the His-P resonance, unlike the one for ATP3'. The assignments of the resonances in these spectra are AMPPCP c~ ( - 1 0 ppm), AMPPCP/3 and 3' (10 and 15 ppm, dependent on Mg2+), coenzyme A phosphomonoester (4 ppm), and phosphodiester ( - 1 0 ppm). (From Vogel and Bridger. 2~)
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
279
glucose increased the mobility for the regulatory phosphoserine-14. 58,59 Although glucose binds to the active site, which is far removed from this regulatory site, it was known that the hydrolysis of the phosphoserine by the protein phosphatase was drastically increased by the glucose addition. 6° Thus 31p NMR provided the rationale for this observation: the binding of glucose to the remote active site increases the mobility and surface exposure of the serine phosphate, allowing the phosphatase to act. One of the problems that can arise when substrates and other ligands are added is that these added components can be phosphorylated. Thus one may run into problems because of overlap of resonances for substrates and phosphoamino acids. Fortunately, these problems can be easily solved because a large number of commercially available analogs for compounds such as ATP, ADP, and AMP have substituted phosphorus groups. As these substitutions bring about characteristic changes in the chemical shift (see Table III), 33,59,6|,62 overlap of resonances can be avoided through a judicious choice of analog (see Fig. 3). The use of these analogs has some further advantages. For example, the bridging methylene and imido substitutions provide for nonhydrolyzable ATP and ADP analogs. Not only does this limit the enzymatic turnover, which would normally complicate the lengthy NMR experiments, but if it can be demonstrated that the analog is a competitive inhibitor, one can also differentiate between events caused by the binding of ATP or phosphorylation by this nucleotide. Also, the slow rate at which thio analogs are used as substrates by many enzymes can be used to advantage, because one can potentially follow the slow turnover. A somewhat different ligand than those discussed above is the electron, which can be introduced into the isoalloxazine ring of the FAD and FMN cofactors of redox proteins. The unpaired electron spin may cause broadening effects on the 31p NMR resonances, which can subsequently be used to estimate the average distance between the electron and the phosphate group. 53-55 Binding of Metal Ions The majority of enzymatic phosphoryl-transfer reactions are strictly dependent on the presence of divalent cations such as Mg2+.63 Not only is 58M. Hoerl, K. Feldmann,K. D. Schnackerz, and E. J. M. Helmreich,Biochemistry 18, 2457 (1979). 59S. G. Withers, N. B. Madsen, and B. D. Sykes,Biochemistry 20, 1748(1981). 6oN. B. Madsen, Enzymes 17, 365 (1986). 61E. K. Jaffe and M. Cohn, Biochemistry 17, 652 (1978). 62S. Tran Dinh and M. Roux, Eur. ]. Biochem. 76, 245 (1977). 63A. S. Mildvan,Adv. Enzymol. 49, 103 (1979).
280
PROTEIN STRUCTURE
[13]
TABLE III 3~p NMR PARAMETERS AND pKa VALUES OF SUBSTITUTED PHOSPHORUS COMPOUNDS
Compound
Change in chemical shifts (ppm) a
Change in pK, b
Thiophosphates
-40
Fluorophosphates
+ 11
No c
Imidophosphates
- 10
+0.9
Methylene phosphates
-25
+ 1.5
9
No c
Cyclic phosphates
-
1,5
Analogs available Nonbridging substitution, slowly hydrolyzable Terminal group of ATP, ADP, and AMP Bridging substitution, nonhydrolyzable Bridging substitution, nonhydrolyzable Nonhydrolyzable, shift depends on bond angle
Reference 61
33
62
33
59
a Negative values indicate a downfield shift. b Negative values indicate a decrease in pKa. c No pH titration occurs between pH 3 and 10; both the fluoro and cyclic phosphate analogs are monoanionic and therefore they do not closely resemble the natural compounds, which are generally mainly dianionic at pH 7.0.
the ATP-Mg 2+ complex (rather than ATP) the preferred substrate, but some of the enzymes are metaUoenzymes and contain tightly bound Z n 2+ and Mg 2+ ions. Thus there has been a considerable interest in using NMR techniques to determine the role of the metal ion. As Zn 2+ does not have useful NMR properties, it has been replaced with H3Cd2+, which is a spin½nucleus. 64 For phosphoglucomutase64 and alkaline phosphatase 65-67 (and w i t h llZCd2+ or 114Cd2+ as a no-spin control), 113Cd-3JP couplings have been observed which provide evidence for the direct coordination of the metal ion to the phosphate group. Unfortunately, however, in our hands this approach has not worked well with enzymes wherein Mg 2+ is not tightly bound to the protein. A second substitution that is often used is the paramagnetic Mn 2+ in place of Mg 2+. Introduction of this cation generally 64 G. I. Rhyu, W. J. Ray, and J. L. Markley, Biochemistry 23, 252 (1984). 65 j. D. Otvos, J. R. Alger, J. E. Coleman, and I. M. Armitage, J. Biol. Chem. 254, 1778 (1979). 66 p. Gettins and J. E. Coleman, J. Biol. Chem. 258, 408 (1983). 67 p. Gettins and J. E. Coleman, J. Biol. Chem. 259, 4991 (1984).
[13]
PHOSPHORUS-31 NMR OF PHOSPHOPROTEINS
281
produces a broadening that can be used to obtain distance information. Mn 2÷, as well as the stable paramagnetic nitroxides, have also been used as general broadening reagents, to determine whether a residue is on the surface or is deeply buried within the protein. The latter residues in general do not experience any line broadening when such agents are added to the solution. If they are on the surface, broadening is generally observed. ~3,55 Conclusions and Other Experiments In the foregoing discussions we have considered how the nature, mobility, and titratability of phosphoproteins can be determined and how these are affected by ligands and metal ions. However, some other types of experiments have also been performed. In the case of the enzyme alkaline phosphatase, both covalent E - P and noncovalent E- P intermediates can be detected in the 3~p NMR spectra. Because these were well separated, it was possible to perform saturation transfer experiments, which allowed the authors to determine the interconversion rates between these catalytic intermediates. 65 Solid-state NMR experiments have also been performed using phosphoproteins. 68These have given support to the idea that there is little difference between the phosphate groups in the protein in solution or in a dried form. Finally, it should be mentioned that the technique of proton detection of nuclei such as t3C and ~SN could also prove useful for studying 31p.69 Unfortunately, however, only in the case of phosphoserine and phosphothreonine can a direct coupling be observed between the proton and phosphorus nuclei; thus it is likely that this technique will remain limited to the study of these two phosphoamino acids. In cases where information is available about the mobility and titratability of the phosphoamino acids, some interesting biochemical generalizations have come forward. Severe restrictions of motions usually occur in the active sites of enzymes. 2 This is consistent with the notion that the immobility facilitates the in-line nucleophilic attack 1° which is known to occur in all phosphoryl transfer enzymes. The fact that most of these sites do not titrate when ligands are bound suggests that these groups are generally shielded from the solvent. Conversely, flexibility and pH titratability were observed for all the sites that were covalently phosphorylated by protein kinasesf1,7°,7~ This flexibility may play an important role allow68 L. J. Banaszak and J. Seelig, Biochemistry 21, 2436 (1982). 69 A. Bax, this series, Vol. 176 [8]. 7o S. P. Williams, W. A. Bridger, and M. N. G. James, Biochemistry 25, 6655 (1986). 7t M. W. Killimann, K. D. Schnackerz, and M. G. Heilmeyer, Biochemistry 23, 112 (1984).
282
PROTEIN STRUCTURE
[14]
ing access of protein kinases and protein phosphatases to the regulatory site. All regulatory phosphorylation sites are dianionic at physiological pH. If salt linkages with basic amino acid side chains occur, the free energy involved TM is AG ° ~< - 5 . 0 kcal mo1-1. However, it is also possible that the highly charged and mobile phosphoryl group would prevent the interaction between two hydrophobic domains on a protein and exert its regulatory function in this fashion. Acknowledgments Research on regulatory proteins in the author's laboratory is presently sponsored by a grant from the Medical Research Council of Canada. The author is the recipient of a Scholarship from the Alberta Heritage Foundation for Medical Research. The secretarial assistance of Susan Clegg is greatly appreciated.
[14] I s o t o p i c L a b e l i n g w i t h H y d r o g e n - 2 a n d C a r b o n - 1 3 to Compare Conformations of Proteins and Mutants G e n e r a t e d b y S i t e - D i r e c t e d M u t a g e n e s i s , II
By JOYCE A. WILDE, PHILIP H. BOLTON, DAVID W. HIBLER, L Y N N HARPOLD, TAYEBEH POURMOTABBED, M A R K D E L L ' A C Q U A , a n d JOHN A . GERLT
Introduction Our preceding chapter in this volume [4] describes the preparation of isotopically labeled staphylococcal nuclease (Snase) samples as well as the characterization of these samples by one-dimensional NMR methods. The one-dimensional NMR spectra clearly show that Snase samples of high isotopic enrichment can be prepared in amounts suitable for NMR studies. In this chapter we describe the strategy for using isotopic labeling to determine the effects of site-specific amino acid replacements on the conformation of wild-type and mutant proteins. A focus of our research has been the investigation of the effects of sitespecific mutations on the activity and conformation of Snase. 1 At the beginning of this project we were confident that active-site mutants of Snase could be generated and that their enzymatic activity could be as1 D. W. Hibler, N. J. Stolowich, M. A. Reynolds, J. A. Gerlt, J. A. Wilde, and P. H. Bolton, Biochemistry 26, 6278 (1987).
METHODS IN ENZYMOLOGY, VOL. 177
Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.