Proc. Natl. Acad. Sci. USA
Vol. 73, No. 5, pp. 1398-1402, May 1976 Chemistry
Nuclear magnetic resonance studies of slowly exchanging peptide protons in cytochrome c in aqueous solution (protein structure/solvent accessibility)
DINSHAW J. PATEL AND LITA L. CANUEL Bell Laboratories, Murray Hill, New Jersey 07974
Communicated by F. A. Bovey, February 11, 1976
ABSTRACT The slowly exchanging protons in oxidized and reduced horse heart cytochrome c (D20, uncorrected pH meter reading 6.5, room temperature) have been monitored by recording the 270 and 360 MHz proton nuclear magnetic resonance spectra of the reduced protein between 5 and 11 parts per million downfield from 2,Aimethyl-2-silapentane-5-sulfonate. There are about 12 well-resolved exchangeable resonances between 7.9 and 10.1 parts per million and the experimental chemical shift data suggest that they originate from the same residues in both oxidation states. These resonances correspond to 19-22 protons in the reduced protein and a lesser number due to partial exchange in the oxidized protein. The base catalysis of the exchange in both oxidation states was used to demonstrate the sequential exchange of these well-resolved resonances in D20. Alternatively, the temperature dependence of the exchange at a fixed pH was used to differentiate and monitor separately the slower and the slowest exchanging resonances as a function of time. The different rates of exchange among the slowly exchanging peptide protons provide evidence against a resistant cluster of protons that exchange together.
the exchange of the remaining labile protons, the exchange being achieved by raising the pH or increasing the temperature (24). The resolution limitations have been overcome by NMR studies at higher frequencies (270 and 360 MHz) and the application of resolution enhancement techniques (25). The slowly exchangeable resonances, designated A to K, between 8 and 10 ppm (Fig. 1) can therefore be monitored and their exchange characteristics can be studied as a function of temperature and pH.
EXPERIMENTAL Sample preparation Horse heart cytochrome c purchased from Sigma Chemical Co. (type III) was used without further purification. The protein was reduced by adding dithionite and the reaction was monitored by optical spectroscopy. Exchange Studies on the Oxidized Protein. Oxidized cytochrome c was dissolved in H20, the pH was adjusted to 6.5, and the solution was lyophilized. The lyophilized material was dissolved in D20 to an 8 mM protein concentration and the pH (uncorrected pH meter reading) was raised to the desired value with NaOD. The samples were heated in a water bath at a given temperature and the aliquots were removed as a function of time. The oxidized samples were reduced with dithionite and the NMR spectra were recorded on the reduced cytochrome c in D20 solution. Exchange Studies on the Reduced Protein. Oxidized cytochrome c was dissolved in H20 solution, the pH was adjusted to 6.5, and the protein was reduced with dithionite. The reduced protein in H20 at pH 6.5 was lyophilized, then dissolved in D20 solution to an 8 mM protein concentration, and the uncorrected pH meter reading was changed to the desired value with NaOD. The samples were heated in the water bath at the desired temperature and aliquots of the reduced protein were removed as a function of time to record the NMR spectra. Proton NMR spectra High resolution proton NMR spectra in D20 were recorded in the Fourier transform mode (repetition time 1.5 sec) on Bruker HX-270 or HX-360 spectrometers that were interfaced with Nicolet BNC-12 computer systems. Water (residual HOD in D20 solutions) was suppressed by applying a low power pulse for 1 sec at the frequency of the HOD resonance prior to applying the ir/2 (12.5 ,tsec for 270 MHz and 15 ,Asec for 360 MHz) pulse (26). Since part of the spectral region consisted of overlapping multiplets, it was necessary to apply resolution enhancement techniques (25). NMR spectra were recorded on 8 mM samples of reduced cytochrome c in DgO solution. The reported pH values are uncorrected pH meter readings (designated pH*). Resonance
Cytochrome c has been studied extensively by chemical and physical techniques with the eventual goal of understanding its role in coupled electron transport (1, 2). X-ray crystallographic studies of the oxidized and reduced cytochrome c have added considerably to our knowledge of the polypeptide conformation and heme group contacts in the crystal (3, 4). Physiological mechanisms have been proposed for the oxidoreduction of cytochrome c based on the available x-ray data on the cytochrome class of proteins (5, 6). Nuclear magnetic resonance (NMR) spectroscopic studies have elucidated many aspects of the cytochrome c -structure in solution (7-12, and references in ref. 9). The specificity of the NMR method can approach and extend the isotopic hydrogen exchange measurements undertaken on proteins (13-15) and applied to the oxidized and reduced states of cytochrome c (16, 17). Slowly exchanging protons have been observed between 7.5 and 11 parts per million (ppm) in the high resolution proton NMR spectrum of bovine pancreatic trypsin inhibitor (18, 19), a highly crosslinked 58 amino acid polypeptide (20), and in oxidized cytochrome and ferredoxin extracted from the thermophilic blue-green alga Synechococcus lividus in 2H20 (i.e., deuterated at the nonexchangeable protons) (21, 22). The exchange characteristics of the peptide protons in lysozyme have been recently reported from Fourier transform nuclear magnetic resonance studies in D20 (difference spectroscopy) and in H20 (by presaturation of the solvent resonance) (23). McDonald and Phillips used proton NMR spectroscopy to demonstrate that about 160 out of the about 200 labile protons in reduced cytochrome c exchange out within 1 hr in D20, pD 5, and room temperature (24). Insufficient resolution at 220 MHz limited the observation of individual resonances during Abbreviations: NMR, nuclear magnetic resonance; ppm, parts per million.
1398
Proc. Natl. Acad. Sci. USA 73 (1976)
Chemistry: Patel and Canuel
Table 1. Chemical shifts and areas of the cytochrome c exchangeable resonances A to M from spectra recorded in Fig. 1
c
Reduced
Reso- Chemical shift, ppm nance A
I0
I 9
1399
8
7
6
V 5
11
7 6 5 9 8 10 FIG. 1. (I) The 360 MHz proton NMR spectrum (5-10 ppm) of ferrocytochrome c in D20, uncorrected pH meter reading 6.95, 270. The reduced protein was dissolved in D20 at pH* 6.5 prior to recording the spectrum. (II) The 360 MHz proton NMR spectrum (5-10 ppm) of ferrocytochrome c in D20, pH* 7.03,27°. The oxidized protein was dissolved in D20 at pH* 6.5; the sample was then reduced and the spectrum was recorded. The exchangeable resonances between 7.8 and 10 ppm are designated by symbols A to M with the nonexchangeable resonance at 9.265 ppm corresponding to one proton.
positions are given as ppm downfield from 2,2-dimethyl-2silapentane-5-sulfonate.
Analysis of data The exchange behavior of the slowly exchanging protons at a given pH* and temperature as a function of time was plotted on a semilog scale representing percent exchange versus time. The meso heme proton at 9.265 ppm was used as an area standard and the exchange behavior of the resonances was monitored by the decrease in their peak heights. For superimposable resonances exhibiting large differences in exchange rates, the separate deuteration behavior could be resolved and the individual exchange rates characterized. RESULTS Spectra The slowly exchanging peptide protons are not well resolved in the NMR spectrum (5-11 ppm) for oxidized cytochrome c, which exhibits contact- and pseudocontact-shifted heme and globin resonances in addition to the aromatic resonances in this spectral region. The reduced protein is diamagnetic and contains aromatic resonances between 5 and 8 ppm, and porphyrin resonances at 9.58 ppm (2 protons), 9.265 ppm (1 proton), and 8.995 ppm (1 proton). The slowly exchanging peptide protons can thus be observed in the spectral window between 8 and 10 ppm. The exchange characteristics of the oxidized protein can be studied by undertaking the experiments on ferricytochrome c and monitoring the results by reducing to ferrocytochrome c and recording the spectra.
A B C D E F G H I J K L M
10.045 9.085 8.995 8.855 8.775 8.675 8.58 8.495 8.36 8.285 8.16 7.955 - 7.84
Oxidized
Area -1 -1 ~-3 -1 ' 1 ~-1 1-2 -2 -2 1-2 3-4
'-1 '-1
Chemical shift, ppm
Area
10.045 9.08 8.995 8.845 8.76 8.67 8.555 8.495 8.345 8.29 8.14 7.98 7.835
Vol. 73, No. 5, pp. 1398-1402, May 1976 Chemistry
Nuclear magnetic resonance studies of slowly exchanging peptide protons in cytochrome c in aqueous solution (protein structure/solvent accessibility)
DINSHAW J. PATEL AND LITA L. CANUEL Bell Laboratories, Murray Hill, New Jersey 07974
Communicated by F. A. Bovey, February 11, 1976
ABSTRACT The slowly exchanging protons in oxidized and reduced horse heart cytochrome c (D20, uncorrected pH meter reading 6.5, room temperature) have been monitored by recording the 270 and 360 MHz proton nuclear magnetic resonance spectra of the reduced protein between 5 and 11 parts per million downfield from 2,Aimethyl-2-silapentane-5-sulfonate. There are about 12 well-resolved exchangeable resonances between 7.9 and 10.1 parts per million and the experimental chemical shift data suggest that they originate from the same residues in both oxidation states. These resonances correspond to 19-22 protons in the reduced protein and a lesser number due to partial exchange in the oxidized protein. The base catalysis of the exchange in both oxidation states was used to demonstrate the sequential exchange of these well-resolved resonances in D20. Alternatively, the temperature dependence of the exchange at a fixed pH was used to differentiate and monitor separately the slower and the slowest exchanging resonances as a function of time. The different rates of exchange among the slowly exchanging peptide protons provide evidence against a resistant cluster of protons that exchange together.
the exchange of the remaining labile protons, the exchange being achieved by raising the pH or increasing the temperature (24). The resolution limitations have been overcome by NMR studies at higher frequencies (270 and 360 MHz) and the application of resolution enhancement techniques (25). The slowly exchangeable resonances, designated A to K, between 8 and 10 ppm (Fig. 1) can therefore be monitored and their exchange characteristics can be studied as a function of temperature and pH.
EXPERIMENTAL Sample preparation Horse heart cytochrome c purchased from Sigma Chemical Co. (type III) was used without further purification. The protein was reduced by adding dithionite and the reaction was monitored by optical spectroscopy. Exchange Studies on the Oxidized Protein. Oxidized cytochrome c was dissolved in H20, the pH was adjusted to 6.5, and the solution was lyophilized. The lyophilized material was dissolved in D20 to an 8 mM protein concentration and the pH (uncorrected pH meter reading) was raised to the desired value with NaOD. The samples were heated in a water bath at a given temperature and the aliquots were removed as a function of time. The oxidized samples were reduced with dithionite and the NMR spectra were recorded on the reduced cytochrome c in D20 solution. Exchange Studies on the Reduced Protein. Oxidized cytochrome c was dissolved in H20 solution, the pH was adjusted to 6.5, and the protein was reduced with dithionite. The reduced protein in H20 at pH 6.5 was lyophilized, then dissolved in D20 solution to an 8 mM protein concentration, and the uncorrected pH meter reading was changed to the desired value with NaOD. The samples were heated in the water bath at the desired temperature and aliquots of the reduced protein were removed as a function of time to record the NMR spectra. Proton NMR spectra High resolution proton NMR spectra in D20 were recorded in the Fourier transform mode (repetition time 1.5 sec) on Bruker HX-270 or HX-360 spectrometers that were interfaced with Nicolet BNC-12 computer systems. Water (residual HOD in D20 solutions) was suppressed by applying a low power pulse for 1 sec at the frequency of the HOD resonance prior to applying the ir/2 (12.5 ,tsec for 270 MHz and 15 ,Asec for 360 MHz) pulse (26). Since part of the spectral region consisted of overlapping multiplets, it was necessary to apply resolution enhancement techniques (25). NMR spectra were recorded on 8 mM samples of reduced cytochrome c in DgO solution. The reported pH values are uncorrected pH meter readings (designated pH*). Resonance
Cytochrome c has been studied extensively by chemical and physical techniques with the eventual goal of understanding its role in coupled electron transport (1, 2). X-ray crystallographic studies of the oxidized and reduced cytochrome c have added considerably to our knowledge of the polypeptide conformation and heme group contacts in the crystal (3, 4). Physiological mechanisms have been proposed for the oxidoreduction of cytochrome c based on the available x-ray data on the cytochrome class of proteins (5, 6). Nuclear magnetic resonance (NMR) spectroscopic studies have elucidated many aspects of the cytochrome c -structure in solution (7-12, and references in ref. 9). The specificity of the NMR method can approach and extend the isotopic hydrogen exchange measurements undertaken on proteins (13-15) and applied to the oxidized and reduced states of cytochrome c (16, 17). Slowly exchanging protons have been observed between 7.5 and 11 parts per million (ppm) in the high resolution proton NMR spectrum of bovine pancreatic trypsin inhibitor (18, 19), a highly crosslinked 58 amino acid polypeptide (20), and in oxidized cytochrome and ferredoxin extracted from the thermophilic blue-green alga Synechococcus lividus in 2H20 (i.e., deuterated at the nonexchangeable protons) (21, 22). The exchange characteristics of the peptide protons in lysozyme have been recently reported from Fourier transform nuclear magnetic resonance studies in D20 (difference spectroscopy) and in H20 (by presaturation of the solvent resonance) (23). McDonald and Phillips used proton NMR spectroscopy to demonstrate that about 160 out of the about 200 labile protons in reduced cytochrome c exchange out within 1 hr in D20, pD 5, and room temperature (24). Insufficient resolution at 220 MHz limited the observation of individual resonances during Abbreviations: NMR, nuclear magnetic resonance; ppm, parts per million.
1398
Proc. Natl. Acad. Sci. USA 73 (1976)
Chemistry: Patel and Canuel
Table 1. Chemical shifts and areas of the cytochrome c exchangeable resonances A to M from spectra recorded in Fig. 1
c
Reduced
Reso- Chemical shift, ppm nance A
I0
I 9
1399
8
7
6
V 5
11
7 6 5 9 8 10 FIG. 1. (I) The 360 MHz proton NMR spectrum (5-10 ppm) of ferrocytochrome c in D20, uncorrected pH meter reading 6.95, 270. The reduced protein was dissolved in D20 at pH* 6.5 prior to recording the spectrum. (II) The 360 MHz proton NMR spectrum (5-10 ppm) of ferrocytochrome c in D20, pH* 7.03,27°. The oxidized protein was dissolved in D20 at pH* 6.5; the sample was then reduced and the spectrum was recorded. The exchangeable resonances between 7.8 and 10 ppm are designated by symbols A to M with the nonexchangeable resonance at 9.265 ppm corresponding to one proton.
positions are given as ppm downfield from 2,2-dimethyl-2silapentane-5-sulfonate.
Analysis of data The exchange behavior of the slowly exchanging protons at a given pH* and temperature as a function of time was plotted on a semilog scale representing percent exchange versus time. The meso heme proton at 9.265 ppm was used as an area standard and the exchange behavior of the resonances was monitored by the decrease in their peak heights. For superimposable resonances exhibiting large differences in exchange rates, the separate deuteration behavior could be resolved and the individual exchange rates characterized. RESULTS Spectra The slowly exchanging peptide protons are not well resolved in the NMR spectrum (5-11 ppm) for oxidized cytochrome c, which exhibits contact- and pseudocontact-shifted heme and globin resonances in addition to the aromatic resonances in this spectral region. The reduced protein is diamagnetic and contains aromatic resonances between 5 and 8 ppm, and porphyrin resonances at 9.58 ppm (2 protons), 9.265 ppm (1 proton), and 8.995 ppm (1 proton). The slowly exchanging peptide protons can thus be observed in the spectral window between 8 and 10 ppm. The exchange characteristics of the oxidized protein can be studied by undertaking the experiments on ferricytochrome c and monitoring the results by reducing to ferrocytochrome c and recording the spectra.
A B C D E F G H I J K L M
10.045 9.085 8.995 8.855 8.775 8.675 8.58 8.495 8.36 8.285 8.16 7.955 - 7.84
Oxidized
Area -1 -1 ~-3 -1 ' 1 ~-1 1-2 -2 -2 1-2 3-4
'-1 '-1
Chemical shift, ppm
Area
10.045 9.08 8.995 8.845 8.76 8.67 8.555 8.495 8.345 8.29 8.14 7.98 7.835
