Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G)

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.     

Open states in native polynucleotides. II. Hydrogen-exchange study of cytosine-containing double helices.

Hydrogen-exchange studies of I · C and G · C double helices were carried out to test the generality of conclusions reached previously in studies of adenine-containing polymers (preceding paper). The cytosine amino group shows hydrogen-exchange behavior similar to the analogous group in adenine; a pH-independent pathway and a parallel general catalysis pathway require prior separation of the base-pair and pre-equilibrium protonation at the ring N. The cytosine amino group does, however, display greater sensitivity to specific and to general catalysis than found for adenine. In the G · C helix, the ring NH proton of guanine exchanges at the opening-limited rate, as does the analogous proton in A · U and A · T pairs, while the guanine amino protons exchange without a prior opening of structure. From the observed exchange rates and the known chemistry for the pH-independent reaction, one can calculate equilibrium opening constants of 4 × 10⁻³ for poly(rI) · poly(rC) and perhaps one tenth of that for poly(rG) · poly(rC). Also the opening rate constant for the G · C helix is 0.01 s⁻¹.These results, when applied to published exchange curves for DNA, indicate an equilibrium opening constant of 0.005, an opening rate constant of 0.04 s⁻¹, and a closing rate constant of 10 s⁻¹. (All values refer to studies at 0 °C.) These values point to the same kind of traveling-loop model for base-pair opening discussed previously for the opening reactions in adenine-containing double helices.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.     

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.     

Spatial structure of gramicidin A in organic solvents. 1H-NMR analysis of conformation heterogeneity in ethanol

Structural features of double helices formed by polypeptides with alternating L- and D-amino acid residues were analysed. It was found that the map of short distances (less than 4 A) between protons of the two backbones is unique for each double helix type and even its fragment implies unambiguously parameters of the helix (i.e. parallel or antiparallel, handedness, pitch of helix, relative shift of polypeptide chains). By analysis of two-dimensional 1H-NMR spectra (COSY, RELSY, HOHAHA, NOESY), proton resonances of [Val1]gramicidin A (GA) in the ethanol solution were assigned. The results obtained show that the solution contains five stable conformations of GA in comparable concentrations. Monomer of GA is in a random coil conformation. Specific maps of short interproton distances for the other four species (1-4) were obtained by means of two dimensional nuclear Overhauser effect spectroscopy. The maps as well as spin-spin couplings of the H-NC alpha-H protons and solvent accessibilities of the individual amide groups correspond to four types of double helices pi pi LD 5,6 with 5.6 residues per turn. The double helices are related to the Veatch species 1-4 of GA. Species 1 and 2 are left-handed parallel double helices increase increase pi pi LD 5,6 with different relative shift of polypeptide chains. Species 3 is a left-handed antiparallel double helix increase decrease pi pi LD 5,6 and species 4 is a right-handed parallel double helix increase increase LD 5,6. In the dimers helices are fixed by the maximum number (28) of interbackbone hydrogen bonds NH...O = C possible for these structures. Species 1, 3 and 4 have C2 symmetry axes. Relationship between gramicidin A spatial structures induced by various media is discussed.     

Helix opening in deoxyribonucleic acid from a proton nuclear magnetic resonance study of imino and amino protons in d(CG)3.

All exchangeable protons in a short DNA helix, d(CG)3 sodium salt, have been studied by proton nuclear magnetic resonance. The cytidine and guanosine amino protons have been assigned for the first time. As a function of temperature the cytidine amino protons and the imino protons behave very similarly, their relaxation is dominated by exchange with solvent above 30 degrees C. The guanosine amino protons, however, show that helix opening can only be described by a multistate model. The most rapid process observed is probably a twist about the helix axis which lengthens or breaks the guanosine amino hydrogen bond and allows rotation of the amino group. The second fastest process is a scissor opening into the major groove which gives rise to solvent exchange with the imino and cytidine amino protons. The slowest process observed is the complete base pair opening in which the guanosine amino protons also exchange with solvent. For the ammonium salt of the oligonucleotide, a specific ammonium ion complex is observed which at low temperature may catalyze exchange of the guanosine amino protons with the protons of the ammonium ion, but retards exchange with solvent. The complex appears to be specific for the sequence d(CpG).     

Amide exchange rates in Escherichia coli acyl carrier protein: correlation with protein structure and dynamics.

The acyl carrier protein (ACP) of Escherichia coli is a 77-amino acid, highly negatively charged three-helix protein that plays a central role in fatty acid biosynthesis. Previous NMR studies have suggested the presence of multiple conformations and marginally stable secondary structural elements. The stability of these elements is now examined by monitoring amide exchange in apo-ACP using NMR-based methods. Because ACP exhibits many rapid exchange rates, application of traditional isotope exchange methods is difficult. In one approach, heteronuclear correlation experiments with pulsed field-gradient coherence selection have reduced the time needed to collect two-dimensional 1H-15N correlation spectra to the point where measurement of exchange of amide protons for deuterium on the timescale of minutes can be made. In another approach, water proton selective inversion-exchange experiments were performed to estimate the exchange rates of protons exchanging on timescales of less than a second. Backbone amide protons in the region of helix II were found to exchange significantly more rapidly than those in helices I and III, consistent with earlier structural models suggesting a dynamic disruption of the second helix. Highly protected amides occur on faces of the helices that may pack into a hydrophobic core present in a partially disrupted state.     

Influence of the polyamines spermine and spermidine on yeast tRNAPhe as revealed from its imino proton NMR spectrum.

A comparison of imino proton NMR spectra of yeast tRNAPhe recorded at various solution conditions indicates, that polyamines have a limited effect on the structure of this tRNA molecule. Polyamines are found to catalyse the solvent exchange of several imino protons in yeast tRNAPhe not only of non hydrogen bonded imino protons, but also of imino protons of the GU and of some AU and tertiary base pairs. It is concluded that at low levels of catalysing components the exchange rates of the latter protons are not determined by the base pair lifetime. In the presence of high levels of spermidine the solvent exchange rates of imino protons of several base pairs in the molecule were assessed as a function of the temperature. Apparent activation energies derived from these rates were found to be less than 80 kJ/mol, which is indicative for (transient) independent opening of the corresponding base pairs. In the acceptor helix the GU base pair acts as a dynamic dislocation. The AU base pairs at one side of the GU base pair exhibit faster transient opening than the GC base pairs on the other side of this wobble pair. The base pairs m2GC10 and GC11 from the D stem and GC28 from the anticodon stem show relatively slow opening up to high temperatures. Model studies suggest that 1-methyladenosine, an element of tRNA itself, catalyses imino proton solvent exchange in a way similar to polyamines.     

The pH dependence of hydrogen-deuterium exchange in trp repressor: the exchange rate of amide protons in proteins reflects tertiary interactions,not only secondary structure.

The pH dependence of amide proton exchange rates have been measured for trp-repressor. One class of protons exchanges too fast to be measured in these experiments. Among the protons that have measurable hydrogen-deuterium exchange rates, two additional classes may be distinguished. The second class of protons are in elements of secondary structure that are mostly on the surface of the protein, and exchange linearly with increasing base concentration (log kex versus pH). The third class of amide protons is characterized by much higher protection against exchange at higher pH. These protons are located in the core of the protein, in helices B and C. The exchange rate in the core region does not increase linearly with pH, but rather goes through a minimum around pH 6. The mechanism of exchange for the slowly exchanging core protons is interpreted in terms of the two-process model of Hilton and Woodward (1979, Biochemistry 18:5834-5841), i.e., exchange through both a local mechanism that does not require unfolding of the protein, and a mechanism involving global unfolding of the protein. The increase in exchange rates at low pH is attributed to a partial unfolding of the repressor. It is concluded that the formation of secondary structure alone is insufficient to account for the high protection factors seen in the core of native proteins at higher pH, and that tertiary interactions are essential to stabilize the structure.     

Base pair opening in three DNA-unwinding elements

DNA-unwinding elements are specific base sequences that are located in the origin of DNA replication where they provide the start point for strand separation and unwinding of the DNA double helix. In the present work we have obtained the first characterization of the opening of individual base pairs in DNA-unwinding elements. The three DNA molecules investigated reproduce the 13-mer DNA-unwinding elements present in the Escherichia coli chromosome. The base sequences of the three 13-mers are conserved in the origins of replication of enteric bacterial chromosomes. The exchange of imino protons with solvent protons was measured for each DNA as a function of the concentration of exchange catalyst using nuclear magnetic resonance spectroscopy. The exchange rates provided the rates and the equilibrium constants for opening of individual base pairs in each DNA at 20 degrees C. The results reveal that the kinetics and energetics of the opening reactions for AT/TA base pairs are different in the three DNA-unwinding elements due to long range effects of the base sequence. These differences encompass the AT/TA base pairs that are conserved in various bacterial genomes. Furthermore, a qualitative correlation is observed between the kinetics and energetics of opening of AT/TA base pairs and the location of the corresponding DNA-unwinding element in the origin of DNA replication.     

Study of amide proton exchange in 15N-enriched cryptogein using pH-dependent off-resonance ROESY-HSQC experiments

A new pH-dependent off-resonance ROESY-HSQC experiment has been used to characterize the degree of protection of the amide protons of cryptogein, a protein of the elicitin family, against solvent exchange. The study of the pH dependence of solvent-shielded amide protons in this protein reveals that the helices have different levels of stability. Two of the five helices exhibit strong protection of amide hydrogens against exchange with the solvent. By contrast, greater flexibility is observed in the other three helices, particularly in the C-terminal helix. These results provide information on the dynamic features of the protein and are consistent with the RMSD for the backbone atoms of residues involved in helical structures. In addition, the question of the flexibility in a hydrophobic cavity made of conserved residues, which represent a plausible binding site, is addressed by this method. Received: 30 July 1997 / Accepted: 11 November 1997     

Periodicity of amide proton exchange rates in a coiled-coil leucine zipper peptide.

The two-stranded coiled-coil motif, which includes leucine zippers, is a simple protein structure that is well suited for studies of helix-helix interactions. The interaction between helices in a coiled coil involves packing of "knobs" into "holes", as predicted by Crick in 1953 and confirmed recently by X-ray crystallography for the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T. (1991) Science 254, 539]. A striking periodicity, extending over six helical turns, is observed in the rates of hydrogen-deuterium exchange for amide protons in a peptide corresponding to the leucine zipper of GCN4. Protons at the hydrophobic interface show the most protection from exchange. The NMR chemical shifts of amide protons in the helices also show a pronounced periodicity which predicts a short H-bond followed by a long H-bond every seven residues. This variation was anticipated in 1953 by Pauling and is sufficient to give rise to a local left-handed superhelical twist characteristic of coiled coils. The amide protons that lie at the base of the "hole" in the "knobs-into-holes" packing show slow amide proton exchange rates and are predicted to have short H-bond lengths. These results suggest that tertiary interactions can lead to highly localized, but substantial, differences in stability and dynamics within a secondary structure element and emphasize the dominant nature of packing interactions in determining protein structure.     

Probing the role of hydrogen bonds in the stability of base pairs in double-helical DNA

Aromatic stacking and hydrogen bonding between nucleobases are two of the key interactions responsible for stabilization of DNA double-helical structures. The present work aims at defining the specific contributions of these interactions to the stability of individual base pairs in DNA. The two DNA double helices investigated are formed, respectively, by the palindromic base sequences 5'-dCCAACGTTGG-3' and 5'-dCGCAGATCTGCG-3'. The strength of the N==H...N inter-base hydrogen bond in each base pair is characterized from the measurement of the protium-deuterium fractionation factor of the corresponding imino proton using NMR spectroscopy. The structural stability of each base pair is evaluated from the exchange rate of the imino proton, measured by NMR. The results reveal that the fractionation factors of the imino protons in the two DNA double helices investigated fall within a narrow range of values, between 0.92 and 1.0. In contrast, the free energies of structural stabilization for individual base pairs span 3.5 kcal/mol, from 5.2 to 8.7 kcal/mol (at 15 degrees C). These findings indicate that, in the two DNA double helices investigated, the strength of N==H...N inter-base hydrogen bonds does not change significantly depending on the nature or the sequence context of the base pair. Hence, the variations in structural stability detected by proton exchange do not involve changes in the strength of inter-base hydrogen bonds. Instead, the results suggest that the energetic identity of a base pair is determined by the number of inter-base hydrogen bonds, and by the stacking interactions with neighboring base pairs.     

Preferential location of bulged guanosine internal to a G.C tract by 1H NMR

A series of double-helical oligodeoxyribonucleotides of sequence corresponding to a frame-shift mutational hot spot in the lambda CI gene, 5'-dGATGGGGCAG, are compared by proton magnetic resonance spectroscopy at 500 MHz of the exchangeable protons. Duplexes containing an extra guanine in a run of two, three, and four G.C base pairs are compared to regular helices of the same sequence and to another sequence containing an isolated bulged G, 5'-dGATGGGCAG.dCTGCGCCATC. The imino proton resonances are assigned by one-dimensional nuclear Overhauser effect spectroscopy. Resonances assigned to the G tract in bulge-containing duplexes are shifted anomalously upfield and are very broad. Imino proton lifetimes are determined by T1 inversion-recovery experiments. The exchange rates of G-tract imino protons in bulged duplexes are rapid compared to those in regular helices and are discussed in terms of the apparent rate of solvent exchange for the isolated G bulge. Delocalization of a bulged guanosine in homopolymeric sequences can explain the observed changes in chemical shift and relaxation times across the entire G.C run, and the chemical shifts can be fit by a simple model of fast exchange between base-paired and unpaired states for the imino protons. This allows us to calculate the relative occupancies of each bulge site. In these sequences, we find the extra base prefers positions internal to the G tract over those at the edge.     

Selective catalysis of A.T base pair proton exchange in DNA complexes: imino proton NMR analysis.

Polyaromatic molecules with amino chain substituents, upon binding with DNA, selectively catalyze exchange of the A.T base pair protons with bulk water protons. The amine-catalyzed exchange is mediated by compounds which are A.T and G.C base sequence specific, intercalators, and outside binders. A mechanism for the selective exchange, involving transient opening and closing of individual A.T base pairs in the duplex, is discussed.     

Spectroscopic studies of bacteriorhodopsin fragments dissolved in organic solution.

Fourier transform infrared and UV fourth-derivative spectroscopies were used to study the secondary structure of bacteriorhodopsin and its chymotryptic and one of the sodium borohydride fragments dissolved in chloroform-methanol (1:1, v/v), 0.1 M LiClO4. The C1 fragment (helices C, D, E, F, and G) showed an alpha-helical content of about 53%, whereas C2 (helices A and B) had about 60%, and B2 (helices F and G) about 65% alpha-helix. The infrared main band indicated differences in alpha-helical properties between these fragments. These techniques were also used to obtain information on the interactions among helices. According to the results obtained from the hydrogen/deuterium exchange kinetics, about 40% of the amide protons of C2 are particularly protected against exchange, whereas for the C1 fragment this process is unexpectedly fast. UV fourth-derivative spectra of these samples were used to obtain information about the environment of Trp side chains. The results showed that the Trp residues of C2 are more shielded from the solvent than those of C1 or B2. The results of this work indicate that the specific interactions existing between the transmembrane segments induce different types of helical conformations in native bacteriorhodopsin.     

Nuclear magnetic spectra of self complementary decanucleotides in solution; base sequence effect on the chemical shifts of nonexchangeable protons

The aim of this study was to attempt to determine the extent to which the chemical shifts of the nonexchangeable base protons of a DNA helix depend upon the base sequence. We measured the proton NMR spectra of twelve decadeoxynucleotides in order to carry out a "statistical" treatment. In the helices, the chemical shifts were found to be determined within +/- 0.04 ppm, largely by the nearest neighbor residues on the 5'-side, and to a smaller extent by the residue on the 3'-side. The theoretical chemical shift calculations reproduced very well the polymerization shifts measured for H2 protons of adenosines if the electrostatic field effect was taken into account. A fair agreement was also obtained for H8 protons of the adenosine and guanosine residues. However, theory underestimates the polarization effects of the base protons of cytidine. This discrepancy suggests that the conformation of this residue is different in the mononucleotides relative to double helices.     

Solution structure and internal dynamics of NSCP, a compact calcium-binding protein

The solution structure of Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) in the calcium-bound form was determined by NMR spectroscopy, distance geometry and simulated annealing. Based on 1859 NOE restraints and 262 angular restraints, 17 structures were generated with a rmsd of 0.87 A from the mean structure. The solution structure, which is highly similar to the structure obtained by X-ray crystallography, includes two open EF-hand domains, which are in close contact through their hydrophobic surfaces. The internal dynamics of the protein backbone were determined by studying amide hydrogen/deuterium exchange rates and 15N nuclear relaxation. The two methods revealed a highly compact and rigid structure, with greatly restricted mobility at the two termini. For most of the amide protons, the free energy of exchange-compatible structural opening is similar to the free energy of structural stability, suggesting that isotope exchange of these protons takes place through global unfolding of the protein. Enhanced conformational flexibility was noted in the unoccupied Ca2+-binding site II, as well as the neighbouring helices. Analysis of the experimental nuclear relaxation and the molecular dynamics simulations give very similar profiles for the backbone generalized order parameter (S2), a parameter related to the amplitude of fast (picosecond to nanosecond) movements of N(H)-H vectors. We also noted a significant correlation between this parameter, the exchange rate, and the crystallographic B factor along the sequence.     

TRANSFORMATION OF NONPOLAR FILAMENTS OF THE BLUE-GREEN ALGA GLOEOTRICHIA ECHINULATA U. WISC. 1052 INTO DOUBLE HELICES12

Normally straight filaments of Gloeotrichia echinulata U. Wisc. 1052 transform into double helices when a critical culture density has been attained. In inorganic Zehnder-Gorham's medium No. 11, the alga initially is morphologically uniform and forms single, lightly curved, polar filaments. When a stage of close proximity of the filaments is reached, excessively long filaments are observed that are nonpolar. Some of these nonpolar filaments form helices. Two independent single helices may entwine to form a double helix. Double helices also form when both ends of an excessively long filament meet and start entwining until a loop is left at about the original middle of the filament. In another manner of double-helix formation, a straight filament makes a hairpin bend at about its middle; then 2 helices form starting at the bend and they entwine into a double helix, leaving a loop at the location of the original bend. Under proper culture conditions, the structures of double helices show an amazing regularity. Once double-helix formation has started, some strong force brings the process to completion. In a mature culture, helices disintegrate into apparently healthy, short pieces of filaments or single cells, while the straight or slightly curved polar filaments still persist. Helical transformation of nonpolar filaments does not appear to be a sign of nutritional or other stress but rather appears to serve a specific purpose. One might speculate that a genetic exchange between opposite cells takes place.