NMR Hydrogen Exchange Research Articles

Atomistic Insights into the Functional Instability of the Second Helix of Fatty Acid Binding Protein

Structural dynamics of fatty acid binding proteins (FABPs), which accommodate poorly soluble ligands in the internalized binding cavities, are intimately related to their function. Recently, local unfolding of the α-helical cap in a variant of human intestinal FABP (IFABP) has been shown to correlate with the kinetics of ligand association, shedding light on the nature of the critical conformational reorganization. Yet, the physical origin and mechanism of the functionally relevant transient unfolding remain elusive. Here, we investigate the intrinsic structural instability of the second helix (αII) of IFABP in comparison with other segments of the protein using hydrogen-exchange NMR spectroscopy, microsecond molecular dynamics simulations, and enhanced sampling techniques. Although tertiary interactions positively contribute to the stability of helices in IFABP, the intrinsic unfolding tendency of αII is encoded in its primary sequence and can be described by the Lifson-Roig theory in the absence of tertiary interactions. The unfolding pathway of αII in intact proteins involves an on-pathway intermediate state that is characterized with the fraying of the last helical turn, captured by independent enhanced sampling methods. The simulations in this work, combined with hydrogen-exchange NMR data, provide new, to our knowledge, atomistic insights into the functional local unfolding of FABPs.

Measuring Solvent Hydrogen Exchange Rates by Multifrequency Excitation 15N CEST: Application to Protein Phase Separation.

Solvent exchange rates provide important information about the structural and dynamical properties of biomolecules. A large number of NMR experiments have been developed to measure such rates in proteins, the great majority of which quantify the buildup of signals from backbone amides after initial perturbation of water magnetization. Here we present a different approach that circumvents the main limitations that result from these classical hydrogen exchange NMR experiments. Building on recent developments that enable rapid recording of chemical exchange saturation transfer (CEST) pseudo-3D data sets, we describe a 15N-based CEST scheme for measurement of solvent exchange in proteins that exploits the one-bond 15N deuterium isotope shift. The utility of the approach is verified with an application to a 236 residue intrinsically disordered protein domain under conditions where it phase separates and a second application involving a mutated form of the domain that does not phase separate, establishing very similar hydrogen exchange rates for both samples. The methodology is well suited for studies of hydrogen exchange in any 15N-labeled biomolecule. A discussion of the merits of the CEST experiment in relation to the popular CLEANEX-PM scheme is presented.

Probing Transient Release of Membrane-Sequestered Tyrosine-Based Signaling Motif by Solution NMR Spectroscopy.

Membrane sequestration of tyrosine-based signaling motifs of antigen receptors effectively restricts the signaling activities in resting lymphocytes. However, low level of basal signaling in resting cells is required for lymphocyte survival and antigen responsiveness, of which the molecular mechanism remains obscure. Here we probe the transient release of the cytoplasmic domain of the membrane-bound IgG heavy chain (mIgG-tail) by hydrogen exchange NMR spectroscopy, illustrating a dynamic molecular basis for its basal signaling activity. To solve the severe resonance overlap problem in the 2D spectra of mIgG-tail, a non-uniformly sampled pseudo-4D hydrogen exchange NMR experiment has been exploited to quantitatively measure site-specific hydrogen exchange rates. Our solution NMR study reveals transient solvent exposure of the ITT signaling motif that can be further enhanced by calcium ion, and provides insight into the mechanism of lymphocyte basal signaling.

Monomeric Potassium Channels may Exist as Heterogeneous Ensemble before Assembly

For many potassium channels, tetramerization domains in either N- or C-terminal of the pore domain assist in tetramer assembly by increasing local concentrations of monomeric subunits. However, the structure of the pore domain as a monomer before tetramerization is not clear. Recently, thiol-labeling experiments have shown that isolated monomers engineered from the voltage-gated potassium channel Kv1.3 can readily adopt their native fold as they are being synthesized. Mutations at certain critical positions make the p-helix become more solvent-exposed; however, the mechanism by how the p-helix becomes more solvent-exposed is unclear. Here, our MD simulations and NMR measurements to show that the monomeric pore domains of potassium channels are heterogeneous and dynamic. According to the simulations, WT Kv1.2 monomer is unstable in its native-like structure and the 3 helices are very dynamic in the membrane. While the 3 helices separate from each other and form many non-native interactions, all helices retain their helical structure and remain buried in the membrane. Simulations of mutants (W384A/W385A/V387E, V387E, and V388E) also show the separation of all 3 helices, yet the p-helix loses helicity and becomes less stable, leading to higher solvent-exposure of the p-helix. We found that the helicity and membrane burial of the p-helix depends on the locations of the inserted charged groups. When a charged group is introduced to the opposite face of the helix as the native aspartate D381, the p-helix leaves the membrane and loses its helicity but when the mutation is on the same face, the helix goes into the interfacial region and retains its helical nature. In addition, our NMR spectroscopy and hydrogen exchange measurements suggest that the WT monomer of KcsA in lipid bicelles may be highly heterogeneous, but are all buried in the membrane. Together, we suggest that the WT monomers are highly dynamic before tetramerization and the locations of mutations on the p-helix highly dictate its helicity and burial.

Quenched Hydrogen Exchange NMR of Amyloid Fibrils.

Amyloid fibrils are associated with a number of human diseases. These aggregatively misfolded intermolecular β-sheet assemblies constitute some of the most challenging targets in structural biology because to their complexity, size, and insolubility. Here, protocols and controls are described for experiments designed to study hydrogen-bonding in amyloid fibrils indirectly, by transferring information about amide proton occupancy in the fibrils to the dimethyl sulfoxide-denatured state. Since the denatured state is amenable to solution NMR spectroscopy, the method can provide residue-level-resolution data on hydrogen exchange for the monomers that make up the fibrils.

Domain-Swapped Dimer of Pseudomonas aeruginosa Cytochrome c551: Structural Insights into Domain Swapping of Cytochrome c Family Proteins

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c 551 (PA cyt c 551), and Hydrogenobacter thermophilus cytochrome c 552 (HT cyt c 552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c 552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c 551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c 551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

Temperature-dependent Kinetics Study for Hydrogen Exchange of Type I Antifreeze Protein from Winter Flounder

C) is not same with their biologically activetemperature (subzero degree). Because aqueous solutioncould be frozen at subzero degree, it is required that thesystematic NMR experiments should be carried out at vari-ous temperatures to understand physical properties of AFPat subzero degree. Here, to further understand the correlationbetween the dynamic properties and antifreeze activities oftype I AFPs, we performed NMR hydrogen exchange experi-ments on the winter flounder type I AFP (Fig. 1, referred toas wt-AFP) at temperatures from 1-23 °C.The winter flounder type I AFP and its mutants, A17L-and A20L-AFPs, were purchased from Cosmo Genetech Inc.(Seoul, Korea) and desalted using a Sephadex G-25 gelfiltration column. Each protein sample (5 mg) was dissolvedin 500 L of 90% H

Multiscale evaluation of pore curvature effects on protein structure in nanopores.

Protein structure in nanopores is an important determinant in porous substrate utilization in biotechnology and materials science. To date, accurate residue details of pore curvature induced protein binding and unfolding were still unknown. Here, a multiscale ensemble of chromatography, NMR hydrogen and deuterium (H/D) exchange, confocal scanning and molecular docking simulations was combined to obtain the protein adsorption information induced by pore size and curvature. Lysozyme and polystyrene microspheres within pores in the 14-120 nm range were utilized as models. With pore size increasing, the bound lysozyme presented a tendency of significantly decreased retention, less unfolding and fewer interacted sites. However, such a significant dependence between pore curvature and protein size only existed in a limited micro-pore range comparable to protein sizes. The mechanism behind the above events could be attributed to the diverse protein interaction area determined by pore curvature and size change, by models calculating the binding of lysozyme onto surfaces. Another surface of opposite curvature for nanoparticles was also calculated and compared, the rules were similar but with opposite direction and such a critical size also existed. These studies of proteins on curved interfaces may ultimately help to guide the design of novel porous materials and assist in the discrimination of the target protein from molecular banks.

Hydrogen Exchange Study of Winter Flounder Type I Antifreeze Protein

Antifreeze proteins (AFPs) are found in various organisms, such as fish, insects, plants, bacteria, and fungi, to promote survival at subzero temperatures by decreasing the freezing point of bodily fluids. AFPs, which are isolated from Arctic and Antarctic fish, comprise several structurally diverse classes of proteins and four classes of structurally independent proteins are identified: type I AFPs, alanine-rich αhelical proteins of 3.3 to 4.5 kDa; type II AFPs, cysteinerich globular proteins containing five disulfide bonds; type III AFPs, 6-kDa globular proteins; and Type IV AFPs, glutamateand glutamine-rich α-helical proteins. The type I AFPs are alanine-rich amphiphilic α-helical proteins found in winter flounder, yellowtail flounder, Alaskan plaice, shorthorn sculpin, and Arctic sculpin. The winter flounder AFP isoform, HPLC6, which is the most extensively studied AFP, contains three 11-amino acid repeats of the sequence Thr-X2-Asx-X7, where X is generally alanine. X-ray crystallographic studies revealed that the type I AFP is completely α-helical in conformation, with the exception of the last residue (R37), which adopts a 310helix. The hydroxyl and methyl groups of four threonine residues, particularly of the central two residues, T13 and T24, play key roles in the ice-binding properties of the type I AFP. For example, T13S/T24S mutations of the type I AFP caused a 90-100% loss in activity relative to wild-type AFP. The mutants, A17L and A21L, where the substitution lies adjacent to the Thr-rich face, caused a significant decrement of the thermal hysteresis activity, whereas the mutants A19L and A20L exhibited wild-type activity. It was reported that termini of the HPLC6 isoform possess greater helix-stabilizing ability compared to the HPLC2 isoform, leading to a 50% difference in the thermal hysteresis activity between these two AFPs. These results suggested that helical stabilization of the N and C termini is the critical component for antifreeze activity of the type I AFP. It was recently reported that flexibility of the C-terminal region causes a loss of thermal hysteresis activity because its dynamic nature strongly prevents binding to the ice surface. Here, to further understand the correlation between the dynamic properties and function of type I AFPs, we performed NMR hydrogen exchange experiments on the winter flounder type I AFP (HPLC6 isoform) (Fig. 1, referred to as wt-AFP). The hydrogen exchange rate constants for the two mutants, A17Land A20L-AFP (Fig. 1), which exhibit no and wild-type activities in thermal hysteresis respectively, are also determined. Comparison of the wtAFP and mutant AFPs can provide valuable insights into the thermal hysteresis mechanism of type I AFPs.

Amide Proton Solvent Protection in Amylin Fibrils Probed by Quenched Hydrogen Exchange NMR

Amylin is an endocrine hormone that accumulates in amyloid plaques in patients with advanced type 2 diabetes. The amyloid plaques have been implicated in the destruction of pancreatic β-cells, which synthesize amylin and insulin. To better characterize the secondary structure of amylin in amyloid fibrils we assigned the NMR spectrum of the unfolded state in 95% DMSO and used a quenched hydrogen-deuterium exchange technique to look at amide proton solvent protection in the fibrils. In this technique, partially exchanged fibrils are dissolved in 95% DMSO and information about amide proton occupancy in the fibrils is determined from DMSO-denatured monomers. Hydrogen exchange lifetimes at pH 7.6 and 37°C vary between ∼5 h for the unstructured N-terminus to 600 h for amide protons in the two β-strands that form inter-molecular hydrogen bonds between amylin monomers along the length of the fibril. Based on the protection data we conclude that residues A8-H18 and I26-Y37 comprise the two β-strands in amylin fibrils. There is variation in protection within the β-strands, particularly for strand β1 where only residues F15-H18 are strongly protected. Differences in protection appear to be due to restrictions on backbone dynamics imposed by the packing of two-layers of C2-symmetry-related β-hairpins in the protofilament structure, with strand β1 positioned on the surface and β2 in the interior.

The C-terminal sterile alpha motif (SAM) domain of human p73 is a highly dynamic protein, which acquires high thermal stability through a decrease in backbone flexibility

The α-splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is involved in protein-biomolecule interactions. The conformational stability of SAMp73 is low (∼5 kcal mol(-1)), although its thermal stability is high. To explain this high thermostability, we studied the dynamics of SAMp73 over a wide range of GdmCl (guanidine hydrochloride) concentrations and temperatures by NMR relaxation, NMR hydrogen-exchange (HX) and fluorescence lifetime approaches. The slowest exchanging residues of SAMp73 belong to the helical regions, and they did exchange by a global unfolding process. Moreover, SAMp73 was very flexible, with most of its amide protons affected by slow μs-ms conformational exchange. Within this time scale, the residues of SAMp73 with the largest exchange rates (R(ex)) were involved in binding with other molecules; therefore, the flexibility in the μs-ms range was associated with biological functions. As the [GdmCl] increased, the pico-to-nanosecond flexibility of the backbone amide protons raised, but it did so differently depending on the residue. We were able to obtain, for the first time, the linear [GdmCl]-variation of the local conformational entropies, m(S(i)), which ranged from 5.3 to 0.3 cal mol(-1) K(-1) M(-1), similar to those measured by using macroscopic techniques in other proteins. Conversely, the temperature dependence of the pico-to-nanosecond dynamics of the backbone amide protons of SAMp73 indicates that the flexibility of some residues decreased with the temperature; these results explain the high thermostability of the protein.

Base pair opening kinetics study of the aegPNA:DNA hydrid duplex containing a site-specific GNA-like chiral PNA monomer

Peptide nucleic acids (PNA) are one of the most widely used synthetic DNA mimics where the four bases are attached to a N-(2-aminoethyl)glycine (aeg) backbone instead of the negative-charged phosphate backbone in DNA. We have developed a chimeric PNA (chiPNA), in which a chiral GNA-like γ3T monomer is incorporated into aegPNA backbone. The base pair opening kinetics of the aegPNA:DNA and chiPNA:DNA hybrid duplexes were studied by NMR hydrogen exchange experiments. This study revealed that the aegPNA:DNA hybrid is much more stable duplex and is less dynamic compared to DNA duplex, meaning that base pairs are opened and reclosed much more slowly. The site-specific incorporation of γ3T monomer in the aegPNA:DNA hybrid can destabilize a specific base pair and its neighbors, maintaining the thermal stabilities and dynamic properties of other base pairs. Our hydrogen exchange study firstly revealed the unique kinetic features of base pairs in the aegPNA:DNA and chiPNA:DNA hybrids, which will provide an insight into the development of methodology for specific DNA recognition using PNA fragments.

Population Analysis of the Intermediate Complex States During B-Z Transition of Non-CG-repeat DNA Duplexes Induced by the Zα Domain of Human ADAR1

Figure 2. (A) The kex values of the G4b imino proton for the CA6ZαADAR1 complex determined at 25 C and (B) kex values of the G2b imino proton for the TA6-ZαADAR1 complex determined at 15 C as a function of the fZ. Black solid lines are the best fit to Eq. 1, where the kex data were weighted by the inverse of their variance. The grey lines indicate their upper and lower confidence limits (95% confidence level). Z-DNA contains nucleic acid bases in alternating antiand syn-conformations along the nucleotide chain and has only one groove that is similar to the minor groove of B-DNA. Z-DNA is in a higher energy conformation than B-DNA and is stabilized by negative supercoiling generated in vivo. Human ADAR1 has two left-handed Z-DNA binding domains at its NH2-terminus, Zα and Zβ, preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The co-crystal structure of the Zα domain of human ADAR1 (ZαADAR1) bound to Z-DNA revealed that one monomeric ZαADAR1 domain binds to one strand of double-stranded DNA and a second ZαADAR1 monomer binds to the opposite strand with two-fold symmetry with respect to the DNA helical axis. A structural study showed that ZαADAR1 binds to the Z-conformation of non-CG-repeat DNA duplexes through a common structural feature rather than by a specific sequence or structural alternations. A previous NMR study on a d(CGCGCG)2-ZαADAR1 complex suggests an active-mono B-Z transition mechanism (see Fig. 1) in which the ZαADAR1 protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second ZαADAR1 molecule. Recently, we have reported NMR hydrogen exchange data of complexes between ZαADAR1 and the non-CG-repeat DNA duplexes, d(CACGTG)2 [referred to as CA6] or d(CGTACG)2 [referred to as TA6], with a variety of protein-to-DNA (P/N) molar ratios. The kex data for the G4b of the CA6-ZαADAR1 complex and for the G2b of the TA6-ZαADAR1 complex showed significant changes as the Z-DNA fraction (fZ) was increased (meaning that the P/N ratio increased) (see Fig. 2). These changes of the kex data can be explained by the presence of mixtures of two imino protons from B-form DNA (referred to as B) and B-DNA-ZαADAR1 complex (referred to as BP) in the imino peaks as given by Eq. 1:

NMR study of hydrogen exchange during the B–Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

The Yaba-like disease viruses (YLDV) are members of the Yatapoxvirus family and have double-stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZα E3L) bound to Z-DNA revealed that the overall structure of yabZα E3L and its interaction with Z-DNA are very similar to those of hZα ADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZα E3L and d(CGCGCG) 2 with a variety of protein-to-DNA molar ratios. This study revealed that yabZα E3L could efficiently change the B-form helix of the d(CGCGCG) 2 to left-handed Z-DNA via the active-mono B–Z transition pathway like hZα ADAR1.

Base pair opening kinetics and dynamics in the DNA duplexes that specifically recognized by very short patch repair protein (Vsr)

In Escherichia coli, the very short patch (VSP) repair system is a major pathway for removal of T·G mismatches in Dcm target sequences. In the VSP repair pathway, the very short patch repair (Vsr) endonuclease selectively recognizes a T·G mismatch in Dcm target sequences and hydrolyzes the 5′-phosphate group of the mismatched thymine. The hydrogen exchange NMR studies here revealed that the T5·G18 mismatch in the Dcm target sequence significantly stabilizes own base pair but destabilizes the two neighboring G4·C19 and A6·T17 base pairs compare to other T·G mismatches. These unusual patterns of base pair stability in the Dcm target sequence can explain how the Vsr endonuclease specifically recognizes the mismatched Dcm target sequence and intercalates into the DNA.

The Structural Characteristics of Nonspecific Lipid Transfer Proteins Explain Their Resistance to Gastroduodenal Proteolysis

The structure and stability of the allergenic nonspecific lipid transfer protein (LTP) of peach were compared with the homologous LTP1 of barley and its liganded form LTP1b. All three proteins were resistant to gastric pepsinolysis and were only slowly digested at 1 to 2 out of 14 potential tryptic and chymotryptic cleavage sites under duodenal conditions. Peach LTP was initially cleaved at Tyr79-Lys80 and then at Arg39-Thr40 (a site lost in barley LTP1). Molecular dynamics simulations of the proteins under folded conditions showed that the backbone flexibility is limited, explaining the resistance to duodenal proteolysis. Arg39 and Lys80 side chains were more flexible in simulations of peach compared with barley LTP1. This may explain differences in the rates of cleavage observed experimentally for the two proteins and suggests that the flexibility of individual amino acid side chains could be important in determining preferred proteolytic cleavage sites. In order to understand resistance to pepsinolysis, proteins were characterized by NMR spectroscopy at pH 1.8. This showed that the helical regions of both proteins remain folded at this pH. NMR hydrogen exchange studies confirmed the rigidity of the structures at acidic pH, with barley LTP1 showing some regions with greater protection. Collectively, these data suggest that the rigidity of the LTP scaffold is responsible for their resistance to proteolysis. Gastroduodenal digestion conditions do not disrupt the 3D structure of peach LTP, explaining why LTPs retain their ability to bind IgE after digestion and hence their allergenic potential.

NMR Conformational and Dynamic Consequences of a Gly to Ser Substitution in an Osteogenesis Imperfecta Collagen Model Peptide

Close packing of three chains in a standard collagen triple helix requires Gly as every third residue. Missense mutations replacing one Gly by a larger residue in the tripeptide repeating sequence in type I collagen are common molecular causes of osteogenesis imperfecta. The structural and dynamic consequences of such mutations are addressed here by NMR studies on a peptide with a Gly-to-Ser substitution within an alpha1(I) sequence. Distances derived from nuclear Overhauser effects indicate that the three Ser residues are still packed in the center of the triple helix and that the standard 1-residue stagger is maintained. NMR dynamics using H-exchange and temperature-dependent amide chemical shifts indicate a greater disruption of hydrogen bonding and/or increased conformational flexibility C-terminal to the Ser site when compared with N terminal. This is consistent with recent suggestions relating clinical severity with an asymmetric effect of residues N- versus C-terminal to a mutation site. Dynamic studies also indicate that the relative position between a Gly in one chain and the mutation site in a neighboring staggered chain influences the disruption of the standard hydrogen-bonding pattern. The structural and dynamic alterations reported here may play a role in the etiology of osteogenesis imperfecta by affecting collagen secretion or interactions with other matrix molecules.

Relative Stabilities of Conserved and Non-Conserved Structures in the OB-Fold Superfamily

The OB-fold is a diverse structure superfamily based on a β-barrel motif that is often supplemented with additional non-conserved secondary structures. Previous deletion mutagenesis and NMR hydrogen exchange studies of three OB-fold proteins showed that the structural stabilities of sites within the conserved β-barrels were larger than sites in non-conserved segments. In this work we examined a database of 80 representative domain structures currently classified as OB-folds, to establish the basis of this effect. Residue-specific values were obtained for the number of Cα-Cα distance contacts, sequence hydrophobicities, crystallographic B-factors, and theoretical B-factors calculated from a Gaussian Network Model. All four parameters point to a larger average flexibility for the non-conserved structures compared to the conserved β-barrels. The theoretical B-factors and contact densities show the highest sensitivity. Our results suggest a model of protein structure evolution in which novel structural features develop at the periphery of conserved motifs. Core residues are more resistant to structural changes during evolution since their substitution would disrupt a larger number of interactions. Similar factors are likely to account for the differences in stability to unfolding between conserved and non-conserved structures.

Hydrogen Exchange Rates of the DNA Oligomer Duplex, d[CGCGCGCCATAAC]/d[GTTATGGCGCGCG] Determined by NMR Spectroscopy

The measurement of hydrogen exchange rates has been an important tool for studying the macromolecular conformation or dynamics. The imino proton resonances of nucleic acids are good probes of the hydrogen exchange study for studying the dynamics of each base pair. The exchange rates of imino protons depend on not only the base pair stability but also solvent accessibility. Hydrogendeuterium (H/D) exchange method is widely used to measure the exchange rates of slowly exchanging imino protons, whereas the exchange rates of fast-exchanging imino protons can be determined by water magnetization transfer method of NMR. NMR hydrogen exchange experiment can provide the information on the thermodynamics and kinetics for basepair opening in the nucleic acids. Lots of results of these experiments determined in various DNAs, RNAs and DNA-protein complexes were reported. Analysis of the hydrogen exchange of imino protons generally was based in the two-state (open/closed) model for the base pair where hydrogen exchange only occurs from the open state. The opening/closing rate constants and/or equilibrium constant for base-pair opening can often be determined by measuring the exchange as a function of the concentration of external catalysts such as ammonia or TRIS. Left-handed Z-DNA is a higher-energy form of the double helix produced at an alternating CG sequence region and is stabilized by high salt concentration or negative super-coiling. Recently, X-ray crystal structure of the B-Z junction induced by the Z-DNA binding protein, where the alternating CGCGCG exhibited left-handed Z-DNA whereas a nonalternating AT-rich region maintained B-form helix, was reported. They also found the continuous stacking of bases between B-DNA and Z-DNA segments with the breaking of one base pair at the junction. This result indicates that the one base pair at the B-Z junction should be destabilized and then its bases might be extruded to allow the B-Z conformational change at the CGCGCG region. Here, we measured the imino proton exchange rates for a bzDNA-13 DNA duplex formed by an 15-nt bzA-15mer, d[GTCGCGCGCCATAAC] and a 13-nt bzB-13mer, d[GTTATGGCGCGCG] (Fig. 1A) by water magnetization transfer method to understand the dynamic character of base pairs. Experimental Section

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

The thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in the P1 duplex using the base catalysts NH3, or TRIS. These equilibrium constants were also measured for several modified P1 duplexes, and the C-2·G23 base pair was the most stable base pair in all the duplexes. The conserved U-1·G22 base pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base pairs on only one side of the wobble. A 2′-OMe modification on the U-3 residue stabilized its own base pair but had little effect on the neighboring base pairs. Three base pairs, U-1·G22, C-2·G23 and A2·U21 showed unusual equilibrium constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the P1 helix with catalytic core are discussed.