Molecular dynamics simulation of thionated hen egg white lysozyme

Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 3(10)-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom-atom distance bounds and (3)J((H)(N)(H)(α))-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited.     

Quantitative evaluation of the chicken lysozyme epitope in the HyHEL-10 Fab complex: free energies and kinetics.

The hen (chicken) egg-white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL-10 Fab (Fab-10) was investigated by alanine scan mutagenesis. The association rate constants (k(on)) for the HEWL Fab-10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (k(off)) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of k(on) fall within a factor of 2 of the wild-type (WT) HEWL value (1.43+/-0.13 X 10(6)M(-1)s(-1)), while the increases in k(off)more nearly reflect the total change in free energies of the complex (deltadeltaG(D)). The dissociation constants (K(D)) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL.Fab-10 complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and D101A antibody complexes are moderately destabilized (0.7 < deltadeltaG(D)< or = 1.0 kcal/mol). Additional mutations of the "hotspot" residues (Tyr20, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the epsilon-amino group of Lys96, contribute to the stability of the complex. The value of deltadeltaG(D) for the R21A mutant complex is a distinct outlier in the Arg21 replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.     

How do two unrelated antibodies,HyHEL‐10 and F9.13.7, recognize the same epitope of hen egg‐white lysozyme?

The anti-hen egg-white lysozyme (HEWL) antibodies HyHEL-10 and F9.13.7 recognize a common epitope. The structures of the complexes differ, however, in the numbers of electrostatic and hydrogen-bond interactions and in the distributions of contacts between the light and heavy chains. The equilibria and kinetics characterizing the F9.13.7 complex formation were evaluated for both wild-type and mutant derivatives of HEWL to help to understand how the different contacts are effectively used in the complexes with the two antibodies. Three epitope hot spots, Y20, K96, and R73 (destabilization > 4 kcal/mole), were found by alanine scanning mutagenesis. The first two constitute two of the three hot spots in the HyHEL-10 complex. The hot spots of the HyHEL-10 paratope are centered on the HEWL epitope; whereas R73 (HEWL), the only important light-chain-contacting residue, is clearly separated from the other hot spots of the F9.13.7 complex. The larger number of epitope warm plus hot spots found in the F9.13.7 complex compared with that of HyHEL-10 shows that the specificity of the former is greater even though the K(D) value is 20-fold larger. Conservative mutations showed that the specificity enhancement is related to the greater number of functional polar and hydrogen bond interactions in the F9.13.7 complex. Alanine scanning mutagenesis would not have illuminated these distinctions. It is shown that the concept of antigen specificity, as defined by cross-reactivity with natural variant antigens, is flawed by phylogenetic bias, and that specificity can only be defined by the use of unbiased epitopes, which are conveniently accessed by site-directed mutagenesis.     

Curcumin's pre-incubation temperature affects its inhibitory potency toward amyloid fibrillation and fibril-induced cytotoxicity of lysozyme

Background

More than twenty-seven human proteins can fold abnormally to form amyloid deposits associated with a number of degenerative diseases. The research reported here is aimed at exploring the connection between curcumin's thermostability and its inhibitory activity toward the amyloid fibrillation of hen egg-white lysozyme (HEWL).

Methods

ThT fluorescence spectroscopy, equilibrium thermal denaturation analysis, and transmission electron microscopy were employed for structural characterization. MTT reduction and flow cytometric analyses were used to examine cell viability.

Results and conclusion

The addition of thermally pre-treated curcumin was found to attenuate the formation of HEWL fibrils and the observed fibrillation inhibition was dependent upon the pre-incubation temperature of curcumin. Our results also demonstrated that the cytotoxic effects of fibrillar HEWL species on PC 12 and SH-SY5Y cells were decreased and negatively correlated with curcumin's thermostability. Next, an enhanced stability of HEWL was perceived upon the addition of curcumin pre-incubated at lower temperature. Furthermore, we found that the alteration of curcumin's thermostability was associated with its inhibitory potency against HEWL fibrillation.

General significance

We believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.     

An insight into the binding of 6-hydroxyflavone with hen egg white lysozyme: a combined approach of multi-spectroscopic and computational studies

Abstract

The interaction of 6-hydroxyflavone (6HF) with hen egg white lysozyme (HEWL) has been executed using multi-spectroscopic and computational methods. Steady state fluorescence studies indicated that static quenching mechanism is involved in the binding of 6HF with HEWL, which was further supported by excited state lifetime and UV–vis absorption studies. The binding constant (K_b) of the HEWL–6HF complex was observed to be 6.44?±?0.09?×?10⁴ M^?1 at 293?K, which decreases with the increase in temperature. The calculation of the thermodynamic quantities showed that the binding is exothermic in nature with a negative enthalpy change (ΔH = ?11.91?±?1.02?kJ mol^?1) along with a positive entropy change (ΔS = +51.36?±?2.43 J K^?1 mol^?1), and the major forces responsible for the binding are hydrogen bonding and hydrophobic interactions. The possibility of energy transfer from tryptophan (Trp) residue to the 6HF ligand was observed from Fo¨rster’s theory. The inclusion of 6HF within the binding site of HEWL induces some micro-environmental changes around the Trp residues as indicated by synchronous and three-dimensional (3D) fluorescence studies. The changes in secondary structural components of HEWL are observed on binding with 6HF along with a reduction in % α-helical content. Computational studies correlate well with the experimental finding, and the ligand 6HF is found to bind near to Trp 62 and Trp 63 residues of HEWL. Altogether, the present study provides an insight into the interaction dynamics and energetics of the binding of 6HF to HEWL.

Communicated by Ramaswamy H. Sarma     

Effects of hesperidin,a flavanone glycoside interaction on the conformation,stability, and aggregation of lysozyme: multispectroscopic and molecular dynamic simulation studies?

Hesperidin (HESP), a flavanone glycoside, shows high antioxidant properties and posses ability to go through the blood–brain barrier. Therefore, it could be a potential drug molecule against aggregation based diseases such as Alzheimer’s, Parkinson’s, and systemic amyloidoses. In this work, we investigated the potential of HESP to interact with hen egg-white lysozyme (HEWL) monomer and prevent its aggregation. The HESP–HEWL binding studies were performed using a fluorescence quenching technique, molecular docking and molecular dynamics simulations. We found a strong interaction of HESP with the lysozyme monomer (K_a, ~ 5 × 10⁴ M^?1) mainly through hydrogen bonding, water bridges, and hydrophobic interactions. We showed that HESP molecule spanned the highly aggregation prone region (amino acid residues 48-101) of HEWL and prevented its fibrillar aggregation. Further, we found that HESP binding completely inhibited amorphous aggregation of the protein induced by disulfide-reducing agent tries-(2-carboxyethyl) phosphine. Conformational and stability studies as followed by various tertiary and secondary structure probes revealed that HESP binding only marginally affected the lysozyme monomer conformation and increased both stability and reversibility of the protein against thermal denaturation. Future studies should investigate detail effects of HESP on solvent dynamics, structure, and toxicity of various aggregates. The answers to these questions will not only target the basic sciences, but also have application in biomedical and biotechnological sciences.     

Effect of silybin on the fibrillation of hen egg‐white lysozyme

In this study, the fibrillation of hen egg‐white lysozyme (HEWL) in the absence and presence of different concentrations of silybin was studied by thioflavin T spectroscopy, Congo red binding assays, 8‐anilino‐1‐naphthalenesulfonic acid (ANS) fluorescence assay, circular dichroism, and transmission electron microscopy. The experimental results indicated that not only the fibrillation of HEWL at high temperature (65°C) and low pH (pH = 2.0) could be inhibited effectively by silybin but also the inhibition of HEWL by silybin followed a dose‐dependent manner. Molecular docking studies indicated that 2 possible binding modes could be found in the interaction between silybin and HEWL via van der Waals forces and electrostatic forces as well as hydrogen bonding. One of these 2 conformations was directly entered into the cavity of HEWL (binding site I); the other was bound to the surface of HEWL (binding site II). In this way, silybin could not only increase the hydrophobicity of the cavity or the surface of HEWL but also influence the microenvironment of the binding site, which was able to stabilize the structure of HEWL and delay the process of HEWL fibrosis.     

Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca²⁺‐induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca²⁺‐myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on ¹⁵N‐labeled recoverin to probe main chain conformational dynamics. ¹⁵N NMR relaxation data suggest that Ca²⁺‐free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca²⁺ levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca²⁺‐dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca²⁺ saturated extruded state (R): T ? I ? R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τ_exδω < 1). The final step (I ? R) is much slower (τ_exδω > 1). The main chain structure of I is similar in part to the structure of half‐saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ? I may transiently increase the exposure of Ca²⁺‐binding sites to initiate Ca²⁺ binding that drives extrusion of the myristoyl group during I ? R. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π¹ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.     

Effects of ultraviolet modification on the gating energetics of cyclic nucleotide-gated channels

Middendorf et al. (Middendorf, T.R., R.W. Aldrich, and D.A. Baylor. 2000. J. Gen. Physiol. 116:227-252) showed that ultraviolet light decreases the current through cloned cyclic nucleotide-gated channels from bovine retina activated by high concentrations of cGMP. Here we probe the mechanism of the current reduction. The channels' open probability before irradiation, P(o)(0), determined the sign of the change in current amplitude that occurred upon irradiation. UV always decreased the current through channels with high initial open probabilities [P(o)(0) > 0.3]. Manipulations that promoted channel opening antagonized the current reduction by UV. In contrast, UV always increased the current through channels with low initial open probabilities [P(o)(0) < or = 0.02], and the magnitude of the current increase varied inversely with P(o)(0). The dual effects of UV on channel currents and the correlation of both effects with P(o)(0) suggest that the channels contain two distinct classes of UV target residues whose photochemical modification exerts opposing effects on channel gating. We present a simple model based on this idea that accounts quantitatively for the UV effects on the currents and provides estimates for the photochemical quantum yields and free energy costs of modifying the UV targets. Simulations indicate that UV modification may be used to produce and quantify large changes in channel gating energetics in regimes where the associated changes in open probability are not measurable by existing techniques.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

Construction of enzyme-substrate complexes between hen egg-white lysozyme and N-acetyl-D-glucosamine hexamer by systematic conformational search and molecular dynamics simulation

We constructed the complexes between HEWL and (GlcNAc)6 oligomer in order to investigate the amino acid residues related to substrate binding in the productive and nonproductive complexes, and the relationship between the distortion of the GlcNAc residue D and the formation of the productive complexes. We obtained 49 HEWL-(GlcNAc)6 complexes by a systematic conformational search and classified the each one to the three binding modes; left side, center, or right side. Furthermore we performed the molecular dynamics simulation against 20 HEWL-(GlcNAc)6 complexes (8: chair model, 12 : half-chair model) selected from the 49 complexes to investigate the interaction between HEWL and (GlcNAc)6. As results, we confirmed that it is necessary for GlcNAc residue D to be half-chaired form to bind toward the right side to form productive complexes. We found newly that eight amino acid residues interact with the (GlcNAc)6 oligomer, as follows, Arg73, Gly102, Asn103 for GlcNAc residue A; Asn103 for GlcNAc residues B and C; Leu56, Ala107, Val109 for GlcNAc residue D; Ala110 for GlcNAc residue E; and Lys33 for GlcNAc residue F. We also indicated that GlcNAc residue F does not interact with Thr47 and rarely interacts with Phe34 and Asn37.     

Role of the triad N46, S106 and T107 and the surface charges in the determination of the acidic pH optimum of digestive lysozymes from Musca domestica

Structures of digestive lysozymes 1 and 2 from housefly (MdL1 and MdL2) show that S106–T107 delimit a polar pocket around E32 (catalytic acid/base) and N46 contributes to the positioning of D50 (catalytic nucleophile), whereas those residues are replaced by V109–A110 and D48 in the non-digestive lysozyme from hen egg-white (HEWL). Further analyses revealed that MdL1 and MdL2 surfaces are less positively charged than HEWL surface. To verify the relevance of these differences to the acidic pH optimum of digestive lysozymes it was determined that pKas of the catalytic residues of the triple mutant MdL2 (N46D–S106V–T107A) are similar to HEWL pKas and higher than those for MdL2. In agreement, triple mutant MdL2 and HEWL exhibits the same pH optimum upon methylumbelliferylchitotrioside. In addition to that, the introduction of six basic residues on MdL1 surface increased by 1 unit the pH optimum for the activity upon bacterial walls. Thus, the acidic pH optimum for MdL2 and MdL1 activities upon methylumbelliferylchitotrioside is determined by the presence of N46, S106 and T107 in the environment of their catalytic residues, which favors pKas reduction. Conversely, acidic pH optimum upon bacterial walls is determined by a low concentration of positive charges on the MdL2 and MdL1 surfaces.     

Arylacetamide kappa opioid receptor agonists with reduced cytochrome P450 2D6 inhibitory activity

Some kappa opioid receptor agonists of the arylacetamide class, for example, ICI 199441 (1), were found to strongly inhibit the activity of cytochrome P450 2D6 (CYP2D6) (1: CYP2D6 IC50=26 nM). Certain analogs bearing a substituted sulfonylamino group, for example, 13, were discovered to have significantly reduced CYP2D6 inhibitory activity (13: CYP2D6 IC50>10 microM) while displaying high affinity toward the cloned human kappa opioid receptor, good kappa/delta and kappa/mu selectivity, and potent in vitro and in vivo agonist activity.     

High critical temperature above T(g) may contribute to the stability of biological systems

In this study, we characterized the molecular mobility around T(g) in sugars, poly-L-lysine and dry desiccation-tolerant biological systems, using ST-EPR, (1)H-NMR, and FTIR spectroscopy, to understand the nature and composition of biological glasses. Two distinct changes in the temperature dependence of the rotational correlation time (tau(R)) of the spin probe 3-carboxy-proxyl or the second moment (M(2)) were measured in sugars and poly-L-lysine. With heating, the first change was associated with the melting of the glassy state (T(g)). The second change (T(c)), at which tau(R) abruptly decreased over several orders of magnitude, was found to correspond with the so-called cross-over temperature, where the dynamics changed from solid-like to liquid-like. The temperature interval between T(g) and T(c) increased in the order of sucrose < trehalose < raffinose 50 degrees C, implying that the stability above T(g) improved in the same order. These differences in temperature-dependent mobilities above T(g) suggest that proteins rather than sugars play an important role in the intracellular glass formation. The exceptionally high T(c) of intracellular glasses is expected to provide excellent long-term stability to dry organisms, maintaining a slow molecular motion in the cytoplasm even at temperatures far above T(g).     

Polarized fluorescence depletion reports orientation distribution and rotational dynamics of muscle cross-bridges

The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers. Previous FP data from single fluorescent probes were limited to the 2(nd)- and 4(th)-rank order parameters, and , of the probe angular distribution (beta) relative to the fiber axis and , a coefficient describing the extent of rapid probe motions. We applied intense 12-micros polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation beam. PFD provides dynamic information describing the extent of motions on the time scale between the fluorescence lifetime (e.g., 4 ns) and the duration of the photoselection pulse and it potentially supplies information about the probe angular distribution corresponding to order parameters above rank 4. Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of iodoacetamidotetramethylrhodamine and exchanged into rabbit psoas muscle fibers. In active contraction, dynamic motions of the RLC on the PFD time scale were intermediate between those observed in relaxation and rigor. The results indicate that previously observed disorder of the light chain region in contraction can be ascribed principally to dynamic motions on the microsecond time scale.     

Localization of the gene encoding R kappa B (NFRKB), a tissue-specific DNA binding protein, to chromosome 11q24-q25.

Although NF (nuclear factor)-kappa B binds in vitro to several of the kappa B regulatory elements found in cellular and viral genes, another DNA binding protein, R kappa B, also binds to a related variant of the kappa B site that regulates interleukin-2 receptor alpha-chain gene expression, a critical event in T cell activation. Southern blot analysis of a human-mouse somatic cell hybrid panel and in situ hybridization using a fluorescent genomic R kappa B probe have allowed assignment of the R kappa B gene (NFRKB) to 11q24-q25. The NFRKB locus is in close proximity to the chromosomal breakpoint implicated in Ewing sarcoma, but it does not appear to span this region. Nonetheless, NFRKB may be particularly useful as the most telomeric marker thus far assigned to 11q.     

Ester-linked hen egg white lysozyme shows a compact fold in a molecular dynamics simulation - possible causes and sensitivity of experimentally observable quantities to structural changes maintaining this compact fold

Prediction and understanding of the folding and stability of the 3D structure of proteins is still a challenge. The different atomic interactions, such as non polar contacts and hydrogen bonding, are known but their exact relative weights and roles when contributing to protein folding and stability are not identified. Initiated by a previous molecular dynamics simulation of fully ester-linked hen egg white lysozyme (HEWL), which showed a more compact fold of the ester-linked molecule compared to the native one, three variants of this protein are analyzed in the present study. These are 129-residue native HEWL, partly ester-linked HEWL, in which only 34 peptide linkages that are not involved in the helical or β-strand parts of native HEWL were replaced by ester linkages, and fully (126 residues) ester-linked HEWL. Native and partly ester-linked HEWL showed comparable behaviour, whereas fully ester-linked HEWL could not maintain the native secondary structure of HEWL in the simulation and adopted a more compact fold. The conformational changes were analyzed by comparing simulation averaged values of quantities that can be measured by NMR, such as (1)H-(15)N backbone order parameters, residual dipolar couplings, proton-proton NOE distances and (3)J-couplings with the corresponding values derived from experimental NMR data for native HEWL. The information content of the latter appeared to be insufficient to detect the local conformational rearrangements upon esterification of the loop regions of the protein. For fully ester-linked HEWL, a significantly reduced agreement was observed. Upon esterification, the backbone-side chain and side chain-side chain hydrogen-bonding pattern of HEWL changes to maintain its compactness and thus the structural stability of the ester-linked lysozymes.     

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R² = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.