Solvent accessibility in folded proteins. Studies of hydrogen exchange in trypsin.

In a native protein, the exchange of a peptide amide proton with solvent occurs by one of two pathways, either directly from the folded protein, or via unfolding, exchange taking place from the unfolded protein. From the thermal unfolding rate constants, the contribution of unfolding to the over-all kinetics as a function of solvent and temperature has been determined. Exchange involving unfolding of the protein is characterized by a high activation energy, in the range of 50 to 60 Cal per mol. The activiation energy (Eapp) of the rates of exchange directly from the folded protein is approximately 20 to 25 Cal per mol. Because for the proton transfer step, Eapp approximately equal to 20 Cal per mol, the activation energy for any contributing protein conformational process(es) is approximately equal to 0 to 5 Cal per mol. Most, if not all, of the peptide amide protons in a folded protein can exchange directly with solvent without the protein unfolding. The number of "slowly" exchanging protons at a given condition of pH and temperature is not related to a discrete structural unit, but rather to the distribution of observed rates within the broader distribution of actual rates. The large attenuation of hydrogen exchange rates in folded proteins, resulting in a distribution of first order rates over 6 orders of magnitude, is primarily due to the effects of restricted solvent accessibility of labile protons in the three-dimensional structure. Any protein conformational process, such as protein fluctuations, invoked to explain the solvent accessibility must be of low activation energy and attenuated by ethanol and other co-solvents (Woodward, C. K., Ellis, L. M., and Rosenberg, A. (1974) J. Biol. Chem. 250, 440-444).     

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 3. 1H and 31P nuclear-magnetic-resonance studies on the phosphocarrier protein HPr; tyrosine titration and denaturation studies.

The phosphocarrier protein HPr has been investigated by proton nuclear magnetic resonance (NMR) at 270 MHz in order to evaluate structural properties of the whole molecule and its active site. The titration behaviour of the three tyrosines of the HPr protein was analysed by monitoring the chemical shifts of the aromatic proton resonances of these residues as a function of pH. It was found that the HPr protein contains a lot of slowly exchanging NH backbone protons which suggested a relatively rigid secondary structure of the protein molecule itself although it contains no disulfide bridges. The HPr protein shows a sharp reversible denaturation behaviour at alkaline pH values. Between pH 10.8 and 11.1 two C-2 proton resonance peaks for the single histidine residue could be observed together with abrupt changes in the aromatic and aliphatic absorption region of the HPr protein which are due to chemical exchange processes. The NMR spectrum of the HPr protein is only changed a little upon raising the temperature from 14 degrees C to 70 degrees C. At 76 degrees C all resonances in the spectrum broaden and almost disappear. This process is irreversible.     

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.     

Sequential 1H and 15N NMR assignments and secondary structure of a recombinant anti-digoxin antibody VL domain.

A uniformly 15N-labeled recombinant light-chain variable (VL) domain from the anti-digoxin antibody 26-10 has been investigated by heteronuclear two-dimensional (2D) and three-dimensional (3D) NMR spectroscopy. Complementary homonuclear 2D NMR studies of the unlabeled VL domain were also performed. Sequence-specific assignments for 97% of the main-chain and 70% of the side-chain proton resonances have been obtained. Patterns of nuclear Overhauser effects observed in 2D NOESY, 3D NOESY-HSQC, and 3D NOESY-TOCSY-HSQC spectra afford a detailed characterization of the VL domain secondary structure in solution. The observed secondary structure--a nine-stranded antiparallel beta-barrel--corresponds to that observed crystallographically for VL domains involved in quaternary associations. The locations of slowly exchanging amide protons have been discerned from a 2D TOCSY spectrum recorded after dissolving the protein in 2H2O. Strands B, C, E, and F are found to be particularly stable. The possible consequences of these results for domain-domain interactions are discussed.     

Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature

With the use of one-dimensional 1H nuclear magnetic resonance, two-dimensional correlated spectroscopy, and two-dimensional nuclear Overhauser enhancement spectroscopy, the exchange mechanisms for numerous individual amide protons in the basic pancreatic trypsin inhibitor (BPTI) were investigated over a wide range of p2H and temperature. Correlated exchange under an EX1 regime was observed only for the most slowly exchanging protons in the central hydrogen bonds of the antiparallel beta-sheet and only over a narrow range of temperature and p2H, i.e., above ca. 55 degrees C and between p2H 7 and 9, where the opening rates of the structure fluctuations which promote the exchange of these protons are of the order 0.1 min-1. At p2H below 7, the exchange of this most stable group of protons is uncorrelated and is governed by an EX2 mechanism. At p2H above 9, the exchange is also uncorrelated and occurs via either EX2 or EX1 processes promoted by strictly local structure fluctuations. For all other backbone amide protons in BPTI, the exchange was found to be uncorrelated and by an EX2 mechanism under all conditions of p2H and temperature where quantitative measurements could be obtained with the methods used, i.e., for kex approximately less than 5 min-1. From these observations with BPTI it can be concluded that the amide proton exchange in globular proteins is quite generally via EX2 processes, with rare exceptions for measurements with extremely stable protons at high temperature and basic p2H. This emphasizes the need for further development of suitable concepts for the structural interpretation of EX2 amide proton exchange [Wagner, G. (1983) Q. Rev. Biophys. 16, 1-57; Wagner, G., Stassinopoulou, C. I., & Wüthrich, K. (1984) Eur. J. Biochem. 145, 431-436] and for more detailed investigations of the intrinsic exchange rates for solvent-exposed amide protons in the "open" states of a protein [Roder, H., Wagner, G., & Wüthrich, K. (1985) Biochemistry (following paper in this issue)].     

Proton NMR assignments and secondary structure of the snake venom protein echistatin.

The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C alpha H, and side-chain resonances, except for the eta-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from 3JC alpha H-NH coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I beta-turn, a short beta-hairpin, and a short, irregular, antiparallel beta-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel beta-sheet.     

Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5-13C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degrees C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in slow exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters: delta H++ = 28 kcal/mol for both I and III; delta S++ = 42 cal/(mol.K) for I, and delta S++ = 41 cal/(mol.K) for III. The remaining tyrosine (V) has a larger enthalpy and entropy of activation: delta H++ - 36 kcal/mol, delta S++ = 72 cal/(mol.K). Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H nuclear-magnetic-resonance studies of the porcine-pancreatic secretory trypsin inhibitor at 270 MHz

The pancreatic secretory trypsin inhibitor from porcine pancreas has been investigated by high-resolution 1H nuclear magnetic resonance (NMR) at 270 MHz. The presence of a number of slowly exchanging labile protons indicates that the protein is highly globular. Of the two tyrosyl rings, one is free-rotating and solvent-exposed while the other one is hindered in its mobility and buried in the interior of the protein. A lineshape analysis of the temperature dependence of aromatic resonances gave the dynamic parameters for activation of ring mobility. The inhibitor exhibits at least three well-resolved high-field ring-current-shifted methyl resonances. Form II of the inhibitor, that lacks the first four residues, has been compared with the intact form I. No detectable differences were found between the spectra of I and II, which indicates that the presence of the N-terminal tetrapeptide does not appreciably affect the overall conformation of the protein.     

Cross-relaxation bottleneck in water-lysozyme proton magnetization exchange

The proton spin-lattice relaxation parameters in natural and deuterated lysozyme solutions have been measured as a function of temperature (0-50 degrees C). The variation of the apparent magnitudes of the water proton magnetizations in the solutions with temperature indicates that magnetic coupling mixes protein and water proton magnetizations. The results are consistent with an exchange cross-relaxation model (Hills, B. P., Mol Phys 1992, 76, 489-508) in which the cross-relaxation acts between the labile and nonlabile protons, rather than between water and protein protons. Although this cross-relaxation pathway clearly affects the observed magnetization fractions in this protein solution, its influence on the relaxation rates is less apparent.     

Stable intermediate states and high energy barriers in the unfolding of GFP

We present a study of the denaturation of a truncated, cycle3 variant of green fluorescent protein (GFP). Chemical denaturation is used to unfold the protein, with changes in structure being monitored by the green fluorescence, tyrosine fluorescence and far-UV circular dichroism. The results show that the denaturation behaviour of GFP is complex compared to many small proteins: equilibrium is established only very slowly, over the time course of weeks, suggesting that there are high folding/unfolding energy barriers. Unfolding kinetics confirm that the rates of unfolding at low concentrations of denaturant are very low, consistent with the slow establishment of the equilibrium. In addition, we find that GFP significantly populates an intermediate state under equilibrium conditions, which is compact and stable with respect to the unfolded state (m(IU)=4.6 kcal mol(-1) M(-1) and Delta G(IU)=12.5 kcal mol(-1)). The global and local stability of GFP was probed further by measuring the hydrogen/deuterium (H/D) NMR exchange rates of more than 157 assigned amide protons. Analysis at two different values of pH showed that amide protons within the beta-barrel structure exchange at the EX2 limit, consequently, free energies of exchange could be calculated and compared to those obtained from the denaturation-curve studies providing further support for the three-state model and the existence of a stable intermediate state. Analysis reveals that amide protons in beta-strands 7, 8, 9 and 10 have, on average, higher exchange rates than others in the beta-barrel, suggesting that there is greater flexibility in this region of the protein. Forty or so amide protons were found which do not undergo significant exchange even after several months and these are clustered into a core region encompassing most of the beta-strands, at least at one end of the barrel structure. It is likely that these residues play an important role in stabilizing the structure of the intermediate state. The intermediate state observed in the chemical denaturation studies described here, is similar to that observed at pH 4 in other studies.     

Hydrogen exchange kinetics of core peptide protons in Streptomyces subtilisin inhibitor

The hydrogen isotope exchange kinetics of the 10 slowest exchanging resonances in the 1H NMR spectrum of Streptomyces subtilisin inhibitor (SSI) have been determined at pH 7-11 and 30-60 degrees C. These resonances are assigned to peptide amide protons in the beta-sheet core that comprises the extensive protein-protein interface of the tightly bound SSI dimer. The core protons are atypical in that their exchange rates are orders of magnitude slower than those for all other SSI protons. When they do exchange at temperatures greater than 50 degrees C, they do so as a set and with a very high temperature coefficient. The pH dependence of the exchange rate constants is also atypical. Exchange rates are approximately first order in hydroxyl ion dependence at pH less than 8.5 and greater than 9.5 and pH independent between pH 8.5 and 9.5. The pH dependence and temperature dependence of the SSI proton exchange rates are interpreted by the two-process model [Woodward, C. K., & Hilton, B. D. (1980) Biophys. J. 32, 561-575]. The results suggest that in the average solution structure of SSI, an unusual mobility of secondary structural elements at the protein surface is, in a sense, compensated by an unusual rigidity and inaccessibility of the beta-sheet core at the dimer interface.     

Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: use of the INEPT experiment to follow individual amides in detergent-solubilized M13 coat protein

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1H nuclear magnetic resonance (NMR) study [O'Neil, J. D. J., & Sykes, B. D. (1988) Biochemistry 27, 2753-2762], multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow "kinetic sets" containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10(5)-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein we use 15N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiment [Morris, G. A., & Freeman, R. (1979) J. Am. Chem. Soc. 101, 760-762] can be used to transfer magnetization to the 15N nucleus from a coupled proton; when 15N-labeled protonated protein is dissolved in 2H2O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H+ and OH- ions. Base catalysis is significantly more effective, resulting in a characteristic minimum rate in model peptides at pH approximately equal to 3. Rate versus pH profiles have been obtained by using the INEPT experiment for the amides of leucine-14, leucine-41, tyrosine-21, tyrosine-24, and valines-29, -30, -31, and -33 in M13 coat protein. The valine residues exchange most slowly and at very similar rates, showing an apparent 10(6)-fold retardation over poly(DL-alanine). A substantial basic shift in the pH of the minimum rate (up to 1.5 pH units) was also observed for some residues. Possible reasons for the shift include accumulation of catalytic H+ ions at the negatively charged micelle surface or destabilization of the negatively charged transition state of the base-catalyzed reaction by either charge or hydrophobic effects within the micelle. The time-dependent exchange-out experiment is suitable for slow exchange rates (kex), i.e., less than (1-2) x 10(-4) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Static and transient hydrogen-bonding interactions in recombinant desulfatohirudin studied by 1H nuclear magnetic resonance measurements of amide proton exchange rates and pH-dependent chemical shifts

With proton nuclear magnetic resonance spectroscopy at 22 degrees C and pD 4.5, individual exchange rates in the range from 2 X 10(-5) to 1 X 10(-1) min-1 were observed for 23 amide protons in recombinant desulfatohirudin. The remaining 38 backbone amide protons exchange more rapidly than 1 X 10(-1) min-1. All 23 slowly exchanging protons are located in the polypeptide segment from residue 4 to residue 42, which forms a well-defined globular domain. Three different breathing modes of this molecular region are manifested in the exchange data, which appear to be correlated with the location of the three disulfide bonds. Chemical shift changes larger than 0.15 ppm between pH 2.5 and pH 5.0 arising from through-space interactions with carboxyl groups were observed for seven backbone amide protons. Two of these shifts can be explained by hydrogen bonds in the core of the protein, Gly 25 NH-Glu 43 O epsilon and Ser 32 NH-Asp 33 O delta, and two others by intraresidual NH-O epsilon interactions in Glu 61 and Glu 62. The remaining three pH shifts for Glu 35, Cys 39, and Ile 59 imply the existence of transient interactions between the molecular core and the flexible C-terminal segment 49-65, which have so far not been characterized by nuclear Overhauser effects or other conformational constraints.     

The kinetics and thermodynamics of reversible denaturation of crystalline soybean trypsin inhibitor

Crystalline soybean trypsin inhibitor protein undergoes denaturation on heating which is reversed on cooling. In the range of temperature of 35 to 50 degrees C. a solution of the protein consists of a mixture of native and denatured forms in equilibrium with each other. The equilibrium is only slowly established and its final value at any temperature is the same whether a heated, denatured solution of the protein is cooled to the given temperature or whether a fresh solution is raised to that temperature. The kinetics of reversible denaturation of the soybean protein as well as the reversal of denaturation is that of a reversible unimolecular reaction, each process consisting at a given temperature of the same two simultaneous reactions acting in opposite directions. The experimental data on the effect of temperature on the velocity and the equilibrium constants of the opposing reaction were utilized in evaluating the reaction energies and activation energies. The reaction energies for denaturation were found to be as follows:- Change in total heat of reaction DeltaH = 57,000 calories per mole Change in entropy of reaction DeltaS = 180 calories per degree per mole The heat of activation DeltaH(1) (double dagger) for denaturation = 55,000 The heat of activation DeltaH(2) (double dagger) for the reversal of denaturation = -1900 The entropy DeltaS(1) (double dagger) for denaturation = 95 The entropy DeltaS(2) (double dagger) for reversal of denaturation = -84     

Amide proton exchange in human metallothionein-2 measured by nuclear magnetic resonance spectroscopy

In human metallothionein-2, the exchange rate constants of ten amide protons were found to range from 1.7 x 10(-4) to 1 x 10(-1) min-1 at pH 6.3 and 8 degrees C. Most of these slowly exchanging protons could be associated with hydrogen bonds in secondary structure elements of the alpha-domain. Amide proton exchange rates thus present an additional criterion for the structural characterization of different metallothioneins, which could be particularly valuable for comparisons of different homologous protein preparations containing nuclear magnetic resonance-inactive metal ions, where the metal-polypeptide co-ordinative bonds cannot be identified directly.     

Nature of lysozyme-water interactions by proton NMR.

Proton spin-lattice relaxation measurements were performed in 10 mM lysozyme solution as a function of temperature and degree of substitution of solvent H2O with D2O. The results show that in the temperature range from 274 to 323 K, the intermolecular lysozyme proton water proton coupling contributes appreciably to the observed water proton relaxation rate. In this system exchange between water protons and labile protein protons does not dominate the behaviour with temperature of the water-lysozyme intermolecular contribution to the spin-lattice relaxation.     

Heat stabilization produced by protein-protein association. A differential scanning calorimetric study of the heat denaturation of the trypsin-soybean trypsin inhibitor and trypsin-ovomucoid complexes.

The irreversible thermal denaturation of the association complexes of bovine beta-trypsin with soybean trypsin inhibitor or ovomucoid was observed with a differential scanning calorimeter. Association of trypsin with either inhibitor results in increased heat stability. The largest effect is observed with beta-trypsin and soybean trypsin inhibitor. At pH 6.7, first order rate constants (s-1) for denaturation at 72 degrees, determined at a heating rate of 10 degrees per min, are: beta-trypsin, 30 times 10-3; soybean trypsin inhibitor, 9 times 10-3; trypsin-soybean trypsin inhibitor complex, 0.4 times 10-3. Under equivalent conditions, rate constants for ovomucoid and trypsin-ovomucoid complex are 4 times 10-3 and 1 times 10-3 s-1, respectively. These changes in rate correspond to heat stabilization of trypsin equivalent to an increase of 16 and 9 degrees, respectively, in its observed denaturation temperature. Rate constants determined for beta-trypsin and trypsin-soybean trypsin inhibitor complex are independent of heating rate; those for soybean trypsin inhibitor and ovomucoid are a function of heating rate. This suggests that predenaturational conformational alterations may be important steps in the denaturation of the inhibitors. Activation energies for denaturation of the complexes and their components are all similar, averaging 70 kcal per mol. The large activation energies observed suggest that denaturation of the complexes is not rate-limited by their dissociation.     

1H NMR studies of plastocyanin from Scenedesmus obliquus: complete sequence-specific assignment, secondary structure analysis, and global fold

Two-dimensional 1H NMR methods have been used to make sequence-specific resonance assignments for the 97 amino acid residues of the plastocyanin from the green alga Scenedesmus obliquus. Assignments were obtained for all backbone protons and the majority of the side-chain protons. Spin system identification relied heavily on the observation of relayed connectivities to the backbone amide proton. Sequence-specific assignments were made by using the sequential assignment procedure. During this process, an extra valine residue was identified that had not been detected in the original amino acid sequence. Elements of regular secondary structure were identified from characteristic NOE connectivities between backbone protons, 3JHN alpha coupling constant values, and the observation of slowly exchanging amide protons. The protein in solution contains eight beta-strands, one short segment of helix, five reverse turns, and five loops. The beta-strands may be arranged into two beta-sheets on the basis of extensive cross-strand NOE connectivities. The chain-folding topology determined from the NMR experiments is that of a Greek key beta-barrel and is similar to that observed for French bean plastocyanin in solution and poplar plastocyanin in the crystalline state. While the overall structures are similar, several differences in local structure between the S. obliquus and higher plant plastocyanins have been identified.     

Dynamic behavior of the imino protons of the gamma OR3 17mer in H2O solution studied by high-resolution NMR

The imino proton resonances of gamma OR3 17mer in water were observed at 500 MHz with the time-shared Redfield pulse train. All of the 17 imino proton resonances could be assigned specifically to individual base pairs by utilizing the trace of NOE connectivities between the imino and adenine C2H protons and between imino protons themselves. AT1 and 17 showed abnormally high chemical shifts in comparison with the other AT pairs. On raising the temperature, broadening of the signal occurred in a sequential manner from the terminals except for AT10 and AT11, which were broadened at a lower temperature than GC12. The relaxation rates of the imino protons were measured by the inversion recovery method. The rates at higher temperatures represent the exchange rates of the imino protons. From the temperature dependences, activation energies of about 15 kcal/mol for the AT imino protons and 23-26 kcal/mol for the GC imino protons were obtained.