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A method for the observation of selected proton NMR resonances of proteins
JOURNAL
OF MAGNETIC
RESONANCE
68, 168- 17 1 ( 1986)
A Method for the Observation of Selected Proton NMR Resonances of Proteins JOYCE A. W ILDE AND PHILIP H. BOLTON* Department of Chemistry, Wesleyan University, Middletown, Connecticut 0645 7
AND Department of Chemistry, University of Maryland, College Park, Maryland 20742 Received November I, 1985; revised January 8, 1986
The determination of protein structures in aqueoussolution representsa formidable problem in biochemistry and m o lecular biology. Although NMR spectroscopy has the potential for providing structural information through proton chemical shifts, proton-proton couplings, and proton-proton nuclear Overhauser effects, the extent of overlap in the proton spectra, whether one or two dimensional, is often too great to allow detailed analysis. A number of approacheshave been used to allow selective detection of only a particular subsetof protons to easeassignmentand overlap problems (I-4); however, all of these methods have significant shortcomings. W e have devised a solution to this problem which relies upon both the high sensitivity selective observation of the NMR spectral properties of protons directly bonded to 13Cand the use of recombinant plasmids which facilitate the economical preparation of the necessary isotopically labeled protein m o lecules. W e have chosen staphylococcal nuclease, Nase, as our experimental system since the structural genewas recently cloned and sequenced(5). The availability of this gene has permitted the construction of a recombinant plasmid that permits the facile preparation of Nase samples that are labeled with r3C in a specific site of a given a m ino acid; in addition, site directed mutagenesis can be used to generate mutant versions of the enzyme that may prove useful in the assignment of the ‘H NMR resonances of selecteda m ino acid residues.Such isotopically labeled Nase samplescan be studied using NMR techniques that allow observation of only those protons directly bonded to a heteronucleus (neighbor protons) (6, 7); a two-dimensional NMR experiment can be used to observe those protons which are coupled to the neighbor protons (remote protons) (6). Selective detection of the neighbors was performed using the pulse sequence -r-acquisition ‘H: 90”-r/2-180”-~/2I3c. -180”-9O”-90”- -decoupling where T is equal to l/(2’&) and the phase cycling is as described previously (6). Proton detection of neighbor and remote protons was performed via relay transfer spectroscopy using the pulse sequence * Fellow of the Alfred P. Sloan Foundation, 1983-1987. t Fellow of the Alfred P. Sloan Foundation, 1981-1985. 0022-2364186 $3.00 Copyright 63 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
168
169
NOTES
‘H: w:
90”-~/2-1800--r/2-180”-
-t* /2-9O”-90”-
-180”-
-t, /2-90”-r-acquisition decoupling
where T is as defined above, tl is the evolution time, and the phase cycling is as described previously (6); the proton signals are 13Cdecoupled in both frequency domains. If 7 is l/(2’.&) the pulse sequence leads to a two-dimensional map with neighbor protons, those with one-bond coupling to 13Cnuclei, on the diagonal and remote protons, those coupled to the neighbors, appearing as cross peaks; there is no detection of protons through long range, i.e., more than one bond, proton-carbon couplings (6). Thus, our methodology greatly reduces spectral overlap problems, facilitates assignment of resonances,and allows observation of only those protons at sites of interest. In this Note we illustrate the effectiveness of our strategy for spectral simplification of proteins by the selective one- and two-dimensional observation of all of the aromatic protons of the tyrosines present in a m illimolar solution of Nase labeled with L-[3,5-‘3Cz]tyrosine. Spectra of the labeled Nase, 20 mg/ml or about 1.2 m M , were obtained in the presence of 10 m M CaC12 and a three-fold excess of thymidine-3’,5’-bisphosphate (pdTp). The 13CNMR spectrum (not shown) is consistent with the presence of the expected eight isotopically labeled tyrosine residues. The extent of isotopic labeling was estimated to be at least 80% by a comparison of the intensities of the proton NMR signals obtained in normal and zero-quantum-filtered spectra of the labeled Nase which are shown in Fig. 1. The results illustrate the effective suppression of all signals from protons other than those directly coupled to a 13C. The sample was also used to determine the chemical shifts of the remote tyrosine 2 and 6 protons by use of proton detection of relay transfer spectroscopy with the results shown in Fig. 2. This two-dimensional chemical-shift-correlation experiment and the one-dimensional zero-quantum spectrum in Fig. 1 allow the observation of the resonances of all of the aromatic protons of the labeled tyrosine residues without overlap from the signals of any other amino acid. The spectra presented in this report clearly demonstrate the ability to simplify proton spectra of dilute samples of proteins with both high sensitivity and resolution, and we anticipate additional useful application of selective detection to nuclear Overhauser effects. Although the choice of labeled tyrosine for the experiments reported here was based both on accessto labeled material and an establishedmethod for its incorporation into proteins synthesizedby prototrophic strains of Escherichiu cofi (a), the recombinant plasmid described in this report together with auxotrophic strains of E. coli will permit the labeling of Nase with most of the remaining amino acids, with the probable exception of alanine and aspartate which are derived directly from citric acid cycle intermediates by transamination reactions. For example, we recently have prepared a sample of Nase isotopically labeled with [4-‘3C]proline following the transformation of our plasmid into a proline auxotrophic strain. Finally, the pulse sequences used in obtaining the spectra shown in the figures should be applicable to the selective observation of enzyme bound substrates and ligands that are enriched with 13Cor any other suitable heteronucleus. The recombinant plasmid used in this study was constructed by the insertion of a restriction fragment bearing the structural gene for Nase (5) in the unique Bum HI
170
NOTES
I
I 8
I 6
I 4
I 2
0 wm
FIG. 1. The upper spectrum is the carbon-13 decoupled proton spectrum of labeled Nase obtained at 45°C in the presence of a threefold excessof pdTp. The lower spectrum is the zero-quantum-filtered proton spectrum obtained on the same sample and is shown at 25 times the gain of the conventional spectrum. The zero-quantum-filtered spectrum was obtained with carbon- 13 decoupling during acquisition and hence only proton-proton couplings are present. The spectra were obtained using a Varian XL-200 NMR spectrometer configured for proton observation and carbon- 13 decoupling using a 10 mm sample tube.
site of the expression vector pCQV2 (9); this plasmid also carries the gene for a temperature sensitive X repressor. Following thermal denaturation of the repressor, the E. co/i host transformed with this plasmid accumulated 50% of its total cellular protein as the Nase gene product; as a result of the precise details of construction of our plasmid, the Nase so produced has the heptapeptide Met-Asp-Pro-Thr-Val-Tyr-Ser appended to the amino terminal alanine of the crystallographically characterized Nase A. Using the general procedure described by Lu and his co-workers for isotopic labeling of aromatic amino acid residues in proteins synthesized by protrophic strains of E. coli (8), the transformed host was grown at 30°C in a minimal medium containing glucose and all amino acids except L-tyrosine; production of isotopically labeled Nase was accomplished by a rapid increase in the temperature of the medium to 42°C and simultaneous addition of [3,5-r3C2]tyrosine to a final concentration of 0.8 mA4. After three hours at 42°C the cells were harvested, and the labeled Nase was isolated from a cell extract by ion exchange and affinity chromatographies. Sixty-four milligrams (3.8 pmol) of homogeneous 13C-labeled Nase was obtained from 1.3 liters of culture medium containing 200 mg of the labeled L-tyrosine.
171
NOTES n 0
c
n
0
: a
7
wm
FIG. 2. The lower spectrum is the zeroquantum-filtered spectrum of labeled Nase obtained at 35°C with carbon- 13 decoupling during acquisition. The three sets of partially resolved tyrosine 3,5-protons are labeled a, b, c from low to high field. The upper three spectra are slices extracted from the absolute value twodimensional map obtained in a proton detected relay transfer experiment which utilized carbon- 13 decoupling during acquisition. The slices shown are along the Fz axis at the F, frequencies of the n, b, and c signals. The slices contain signals from the neighbor tyrosine 3,5-protons, labeled n, and from remote tyrosine 2,6protons, labeled r, as well as some overlap signals due to incomplete resolution of the data which are labeled o. The spectral width in each dimension was 500 Hz and 64 increments of the evolution time were used. The F, data were zero-filled to 128 points and there were 5 12 points along F2. Line broadening of 2.5 Hz was used in each dimension. For each evolution time 1024 transients were accumulated with an Fz acquisition time of 0.4 s. The total time for the experiment was just under 18 hr. All spectra were obtained using a Varian XL-200 NMR spectrometer configured for proton observation and carbon-13 decoupling during acquisition. The 10 mm sample used to acquire the data in Fig. 1 was used for this experiment.
ACKNOWLEDGMENTS The labeled tyrosine was supplied by the NIH Stable Isotope Research Resource at the Los Alamos National Laboratory (RR-02231) with the generous assistance of Dr. Clifford Unkefer and Dr. Thomas Walker. This research was supported, in part, by Grant PCM-8314322 from the National Science Foundation (to P.H.B.) and Grant GM-34573 from the National Institutes of Health (to J.A.G.). REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9.
J. L. MARKLEY AND 0. JARDETZKY, J. Mol. Biol. 50,223 (I 970). K. WOTHRICH, G. WIDER, G. WAGNER, AND W. BRANN, J. Mol. Biol. 155, 3 11 (1982). M. BILLETER, W. BRANN, AND K. WOTHRICH, J. Mol. Biol. 155,321 (1982). G. WAGNER AND K. WOTHRICH, J. Mol. Biol. 155, 347 (1982). D. SHORTLE, Gene 22, 181 (1983). P. H. BOLTON, J. Magn. Reson. 62, 143 (1985). R. H. GRIF’FEY, A. G. REDFIELD, R. E. LOOMIS, AND F. W. DAHLQUIST, Biochemistry 24,8 17 (1985). P. Lu, M. JAREMA, K. MOSSER, AND W. E. DANIEL, Proc. Natl. Acad. Sci. USA 73, 347 I (1976). C. QUEEN, J. Mol. Appl. Genet. 2, 1, (1983).
OF MAGNETIC
RESONANCE
68, 168- 17 1 ( 1986)
A Method for the Observation of Selected Proton NMR Resonances of Proteins JOYCE A. W ILDE AND PHILIP H. BOLTON* Department of Chemistry, Wesleyan University, Middletown, Connecticut 0645 7
AND Department of Chemistry, University of Maryland, College Park, Maryland 20742 Received November I, 1985; revised January 8, 1986
The determination of protein structures in aqueoussolution representsa formidable problem in biochemistry and m o lecular biology. Although NMR spectroscopy has the potential for providing structural information through proton chemical shifts, proton-proton couplings, and proton-proton nuclear Overhauser effects, the extent of overlap in the proton spectra, whether one or two dimensional, is often too great to allow detailed analysis. A number of approacheshave been used to allow selective detection of only a particular subsetof protons to easeassignmentand overlap problems (I-4); however, all of these methods have significant shortcomings. W e have devised a solution to this problem which relies upon both the high sensitivity selective observation of the NMR spectral properties of protons directly bonded to 13Cand the use of recombinant plasmids which facilitate the economical preparation of the necessary isotopically labeled protein m o lecules. W e have chosen staphylococcal nuclease, Nase, as our experimental system since the structural genewas recently cloned and sequenced(5). The availability of this gene has permitted the construction of a recombinant plasmid that permits the facile preparation of Nase samples that are labeled with r3C in a specific site of a given a m ino acid; in addition, site directed mutagenesis can be used to generate mutant versions of the enzyme that may prove useful in the assignment of the ‘H NMR resonances of selecteda m ino acid residues.Such isotopically labeled Nase samplescan be studied using NMR techniques that allow observation of only those protons directly bonded to a heteronucleus (neighbor protons) (6, 7); a two-dimensional NMR experiment can be used to observe those protons which are coupled to the neighbor protons (remote protons) (6). Selective detection of the neighbors was performed using the pulse sequence -r-acquisition ‘H: 90”-r/2-180”-~/2I3c. -180”-9O”-90”- -decoupling where T is equal to l/(2’&) and the phase cycling is as described previously (6). Proton detection of neighbor and remote protons was performed via relay transfer spectroscopy using the pulse sequence * Fellow of the Alfred P. Sloan Foundation, 1983-1987. t Fellow of the Alfred P. Sloan Foundation, 1981-1985. 0022-2364186 $3.00 Copyright 63 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
168
169
NOTES
‘H: w:
90”-~/2-1800--r/2-180”-
-t* /2-9O”-90”-
-180”-
-t, /2-90”-r-acquisition decoupling
where T is as defined above, tl is the evolution time, and the phase cycling is as described previously (6); the proton signals are 13Cdecoupled in both frequency domains. If 7 is l/(2’.&) the pulse sequence leads to a two-dimensional map with neighbor protons, those with one-bond coupling to 13Cnuclei, on the diagonal and remote protons, those coupled to the neighbors, appearing as cross peaks; there is no detection of protons through long range, i.e., more than one bond, proton-carbon couplings (6). Thus, our methodology greatly reduces spectral overlap problems, facilitates assignment of resonances,and allows observation of only those protons at sites of interest. In this Note we illustrate the effectiveness of our strategy for spectral simplification of proteins by the selective one- and two-dimensional observation of all of the aromatic protons of the tyrosines present in a m illimolar solution of Nase labeled with L-[3,5-‘3Cz]tyrosine. Spectra of the labeled Nase, 20 mg/ml or about 1.2 m M , were obtained in the presence of 10 m M CaC12 and a three-fold excess of thymidine-3’,5’-bisphosphate (pdTp). The 13CNMR spectrum (not shown) is consistent with the presence of the expected eight isotopically labeled tyrosine residues. The extent of isotopic labeling was estimated to be at least 80% by a comparison of the intensities of the proton NMR signals obtained in normal and zero-quantum-filtered spectra of the labeled Nase which are shown in Fig. 1. The results illustrate the effective suppression of all signals from protons other than those directly coupled to a 13C. The sample was also used to determine the chemical shifts of the remote tyrosine 2 and 6 protons by use of proton detection of relay transfer spectroscopy with the results shown in Fig. 2. This two-dimensional chemical-shift-correlation experiment and the one-dimensional zero-quantum spectrum in Fig. 1 allow the observation of the resonances of all of the aromatic protons of the labeled tyrosine residues without overlap from the signals of any other amino acid. The spectra presented in this report clearly demonstrate the ability to simplify proton spectra of dilute samples of proteins with both high sensitivity and resolution, and we anticipate additional useful application of selective detection to nuclear Overhauser effects. Although the choice of labeled tyrosine for the experiments reported here was based both on accessto labeled material and an establishedmethod for its incorporation into proteins synthesizedby prototrophic strains of Escherichiu cofi (a), the recombinant plasmid described in this report together with auxotrophic strains of E. coli will permit the labeling of Nase with most of the remaining amino acids, with the probable exception of alanine and aspartate which are derived directly from citric acid cycle intermediates by transamination reactions. For example, we recently have prepared a sample of Nase isotopically labeled with [4-‘3C]proline following the transformation of our plasmid into a proline auxotrophic strain. Finally, the pulse sequences used in obtaining the spectra shown in the figures should be applicable to the selective observation of enzyme bound substrates and ligands that are enriched with 13Cor any other suitable heteronucleus. The recombinant plasmid used in this study was constructed by the insertion of a restriction fragment bearing the structural gene for Nase (5) in the unique Bum HI
170
NOTES
I
I 8
I 6
I 4
I 2
0 wm
FIG. 1. The upper spectrum is the carbon-13 decoupled proton spectrum of labeled Nase obtained at 45°C in the presence of a threefold excessof pdTp. The lower spectrum is the zero-quantum-filtered proton spectrum obtained on the same sample and is shown at 25 times the gain of the conventional spectrum. The zero-quantum-filtered spectrum was obtained with carbon- 13 decoupling during acquisition and hence only proton-proton couplings are present. The spectra were obtained using a Varian XL-200 NMR spectrometer configured for proton observation and carbon- 13 decoupling using a 10 mm sample tube.
site of the expression vector pCQV2 (9); this plasmid also carries the gene for a temperature sensitive X repressor. Following thermal denaturation of the repressor, the E. co/i host transformed with this plasmid accumulated 50% of its total cellular protein as the Nase gene product; as a result of the precise details of construction of our plasmid, the Nase so produced has the heptapeptide Met-Asp-Pro-Thr-Val-Tyr-Ser appended to the amino terminal alanine of the crystallographically characterized Nase A. Using the general procedure described by Lu and his co-workers for isotopic labeling of aromatic amino acid residues in proteins synthesized by protrophic strains of E. coli (8), the transformed host was grown at 30°C in a minimal medium containing glucose and all amino acids except L-tyrosine; production of isotopically labeled Nase was accomplished by a rapid increase in the temperature of the medium to 42°C and simultaneous addition of [3,5-r3C2]tyrosine to a final concentration of 0.8 mA4. After three hours at 42°C the cells were harvested, and the labeled Nase was isolated from a cell extract by ion exchange and affinity chromatographies. Sixty-four milligrams (3.8 pmol) of homogeneous 13C-labeled Nase was obtained from 1.3 liters of culture medium containing 200 mg of the labeled L-tyrosine.
171
NOTES n 0
c
n
0
: a
7
wm
FIG. 2. The lower spectrum is the zeroquantum-filtered spectrum of labeled Nase obtained at 35°C with carbon- 13 decoupling during acquisition. The three sets of partially resolved tyrosine 3,5-protons are labeled a, b, c from low to high field. The upper three spectra are slices extracted from the absolute value twodimensional map obtained in a proton detected relay transfer experiment which utilized carbon- 13 decoupling during acquisition. The slices shown are along the Fz axis at the F, frequencies of the n, b, and c signals. The slices contain signals from the neighbor tyrosine 3,5-protons, labeled n, and from remote tyrosine 2,6protons, labeled r, as well as some overlap signals due to incomplete resolution of the data which are labeled o. The spectral width in each dimension was 500 Hz and 64 increments of the evolution time were used. The F, data were zero-filled to 128 points and there were 5 12 points along F2. Line broadening of 2.5 Hz was used in each dimension. For each evolution time 1024 transients were accumulated with an Fz acquisition time of 0.4 s. The total time for the experiment was just under 18 hr. All spectra were obtained using a Varian XL-200 NMR spectrometer configured for proton observation and carbon-13 decoupling during acquisition. The 10 mm sample used to acquire the data in Fig. 1 was used for this experiment.
ACKNOWLEDGMENTS The labeled tyrosine was supplied by the NIH Stable Isotope Research Resource at the Los Alamos National Laboratory (RR-02231) with the generous assistance of Dr. Clifford Unkefer and Dr. Thomas Walker. This research was supported, in part, by Grant PCM-8314322 from the National Science Foundation (to P.H.B.) and Grant GM-34573 from the National Institutes of Health (to J.A.G.). REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9.
J. L. MARKLEY AND 0. JARDETZKY, J. Mol. Biol. 50,223 (I 970). K. WOTHRICH, G. WIDER, G. WAGNER, AND W. BRANN, J. Mol. Biol. 155, 3 11 (1982). M. BILLETER, W. BRANN, AND K. WOTHRICH, J. Mol. Biol. 155,321 (1982). G. WAGNER AND K. WOTHRICH, J. Mol. Biol. 155, 347 (1982). D. SHORTLE, Gene 22, 181 (1983). P. H. BOLTON, J. Magn. Reson. 62, 143 (1985). R. H. GRIF’FEY, A. G. REDFIELD, R. E. LOOMIS, AND F. W. DAHLQUIST, Biochemistry 24,8 17 (1985). P. Lu, M. JAREMA, K. MOSSER, AND W. E. DANIEL, Proc. Natl. Acad. Sci. USA 73, 347 I (1976). C. QUEEN, J. Mol. Appl. Genet. 2, 1, (1983).