Heteronuclear Nuclear Overhauser Effect Research Articles

NMR investigations on binding and dynamics of imidazolium-based ionic liquids with HEWL.

Molecular level insights on protein-ionic liquid (P-IL) interactions are beneficial for assessing protein stability, binding and dynamics. In the present work, interactions of ILs, namely, 1-butyl 3-methylimidazolium methyl sulfate (IL1), 1-butyl 3-methylimidazolium octyl sulfate (IL2) and 1-butyl 3-methylimidazolium chloride (IL3) with hen egg white lysozyme (HEWL) protein were investigated using solution-state nuclear magnetic resonance (NMR) spectroscopy. To ascertain the binding and dynamics from the perspective of both protein and IL, various ligand based NMR approaches such as selective and non-selective nuclear spin-relaxation (R1SEL and R1NS), saturation transfer difference (STD), difference of inversion recovery rate with and without target irradiation (DIRECTION), 35Cl line-shape and spin-relaxation, and protein back bone amide chemical shift perturbations (CSPs) from 1H-15N HSQC were utilized. Among the ILs investigated, IL2 experiences significant interaction relative to those of IL1 and IL3, as revealed by the combined R1SEL and R1NS analysis, which is further supported by STD NMR. CSP analyses of 1H-15N HSQC spectra of aqueous P-IL mixtures enabled to identify the potential binding sites of ILs with HEWL. Whereas, 15N longitudinal (R1) and transverse (R2) spin-relaxation rates and 15N{1H} heteronuclear nuclear Overhauser effect (hetNOE) data subjected to the model free analysis for IL2 yielded the rotational correlation times and order parameters of various residues of HEWL. Furthermore, the results could discern the nature of interactions between studied ILs and HEWL in terms of specific and non-specific interactions.

Exploiting heterogeneous time scale of dynamics to enhance 2D HETCOR solid-state NMR sensitivity.

Multidimensional solid-state NMR spectroscopy plays a significant role in offering atomic-level insights into molecular systems. In particular, heteronuclear chemical shift correlation (HETCOR) experiments could provide local chemical and structural information in terms of spatial heteronuclear proximity and through-bond connectivity. In solid state, the transfer of magnetization between heteronuclei, a key step in HETCOR experiments, is usually achieved using cross-polarization (CP) or insensitive nuclei enhanced by polarization transfer (INEPT) depending on the sample characteristics and magic-angle-spinning (MAS) frequency. But, for a multiphase system constituting molecular components that differ in their time scales of mobilities, CP efficiency is pretty low for mobile components because of the averaging of heteronuclear dipolar couplings whereas INEPT is inefficient for immobile components due to the short T2 and can yield through-space connectivity due to strong proton spin diffusion for immobile components especially under moderate spinning speeds. Herein, in this study we present two 2D pulse sequences that enable the sequential acquisition of 13C/1H HETCOR NMR spectra for the rigid and mobile components by taking full advantage of the abundant proton magnetization in a single experiment with barely increasing the overall experimental time. In particular, the 13C-detected HETCOR experiment could be applied under slow MAS conditions, where a multiple-pulse sequence is typically employed to enhance 1H spectral resolution in the indirect dimension. In contrast, the 1H-detected HETCOR experiment should be applied under ultrafast MAS, where CP and heteronuclear nuclear Overhauser effect (NOE) polarization transfer are combined to enhance 13C signal intensities for mobile components. These pulse sequences are experimentally demonstrated on two model systems to obtain 2D 13C/1H chemical shift correlation spectra of rigid and mobile components independently and separately. These pulse sequences can be used for dynamics based spectral editing and resonance assignments. Therefore, we believe the proposed 2D HETCOR NMR pulse sequences will be beneficial for the structural studies of heterogeneous systems containing molecular components that differ in their time scale of motions for understanding the interplay of structures and properties.

Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor Binding Affinity.

The oncoprotein MdmX (mouse double minute X) is highly homologous to Mdm2 (mouse double minute 2) in terms of their amino acid sequences and three-dimensional conformations, but Mdm2 inhibitors exhibit very weak affinity for MdmX, providing an excellent model for exploring how protein conformation distinguishes and alters inhibitor binding. The intrinsic conformation flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of inhibitors. Although the molecular dynamics simulation approach enables us to understand protein-ligand interactions, the mechanism underlying how a flexible binding pocket adapts an inhibitor has been less explored experimentally. In this work, we have investigated how the intrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affect the affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guided by heteronuclear nuclear Overhauser effect measurements, we identified the flexible regions that affect inhibitor binding affinity around the ligand-binding pocket on N-MdmX. A disulfide engineering mutant, N-MdmXC25-C110/C76-C88, which incorporated two staples to rigidify the ligand-binding pocket, allowed an affinity for nutlin-3a higher than that of wild-type N-MdmX (Kd ∼ 0.48 vs Kd ∼ 20.3 μM). Therefore, this mutant provides not only an effective protein model for screening and designing of MdmX inhibitors but also a valuable clue for enhancing the intermolecular interactions of the pharmacophores of a ligand with pronounced flexible regions. In addition, our results revealed an allosteric ligand-binding mechanism of N-MdmX in which the ligand initially interacts with a compact core, followed by augmenting intermolecular interactions with intrinsic flexible regions. This strategy should also be applicable to many other protein targets to accelerate drug discovery.

Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

SummaryThe Notch receptor is a key component of a core metazoan signaling pathway activated by Delta/Serrate/Lag-2 ligands expressed on an adjacent cell. This results in a short-range signal with profound effects on cell-fate determination, cell proliferation, and cell death. Key to understanding receptor function is structural knowledge of the large extracellular portion of Notch which contains multiple repeats of epidermal growth factor (EGF)-like domains. Here we investigate the EGF4-13 region of human Notch1 (hN1) using a multidisciplinary approach. Ca2+-binding measurements, X-ray crystallography, {1H}-15N heteronuclear nuclear Overhauser effects, and residual dipolar couplings support a non-linear organization for the EGF4-13 region with a rigid, bent conformation for EGF4-7 and a single flexible linkage between EGF9 and EGF10. These data allow us to construct an informed model for EGF10-13 which, in conjunction with comparative binding studies, demonstrates that EGF10 has an important role in determining Notch receptor sensitivity to Dll-4.

A new rosane-type diterpenoid from Stachys parviflora and its density functional theory studies

A new rosane-type diterpenoid (1) has been isolated from the chloroform fraction of Stachys parviflora. Structure of 1 was proposed based on 1D and 2D NMR techniques including correlation spectroscopy, heteronuclear multiple quantum coherence, heteronuclear multiple bond correlation and nuclear Overhauser effect spectroscopy. A theoretical model for the electronic and spectroscopic properties of compound 1 is also developed. The geometries and electronic properties were modelled at B3LYP/6-31G* and the theoretical scaled spectroscopic data correlate nicely with the experimental data.

A disordered region in the EvpP protein from the type VI secretion system of Edwardsiella tarda is essential for EvpC binding.

The type VI secretion system (T6SS) of pathogenic bacteria plays important roles in both virulence and inter-bacterial competitions. The effectors of T6SS are presumed to be transported either by attaching to the tip protein or by interacting with HcpI (haemolysin corregulated protein 1). In Edwardsiella tarda PPD130/91, the T6SS secreted protein EvpP (E. tarda virulent protein P) is found to be essential for virulence and directly interacts with EvpC (Hcp-like), suggesting that it could be a potential effector. Using limited protease digestion, nuclear magnetic resonance heteronuclear Nuclear Overhauser Effects, and hydrogen-deuterium exchange mass spectrometry, we confirmed that the dimeric EvpP (40 kDa) contains a substantial proportion (40%) of disordered regions but still maintains an ordered and folded core domain. We show that an N-terminal, 10-kDa, protease-resistant fragment in EvpP connects to a shorter, 4-kDa protease-resistant fragment through a highly flexible region, which is followed by another disordered region at the C-terminus. Within this C-terminal disordered region, residues Pro143 to Ile168 are essential for its interaction with EvpC. Unlike the highly unfolded T3SS effector, which has a lower molecular weight and is maintained in an unfolded conformation with a dedicated chaperone, the T6SS effector seems to be relatively larger, folded but partially disordered and uses HcpI as a chaperone.

Pair dynamics and the intermolecular nuclear Overhauser effect (NOE) in liquids analysed by simulation and model theories: application to an ionic liquid.

Combining simulation and model theories, this paper analyses the impact of pair dynamics on the intermolecular nuclear Overhauser effect (NOE) in liquids. For the first time, we give a distance resolved NOE. When applied to the ionic liquid 1-ethyl-3-methyl-imidazolium tetrafluoroborate the NOE turns out to be of long-range nature. This behaviour translates to the experimentally measured cross- and longitudinal relaxation rates. We were able to calculate the heteronuclear NOE from simulation data, despite the high computational effort. Model theories are computationally less demanding and cover the complete frequency range of the respective spectral density function, they are usually based on a very simple pair distribution function and the solution of the diffusion equation. In order to model the simulated data sufficiently, these simplifications in structure and dynamics have to be generalised considerably.

Relationship between Structural Flexibility and Function in the C-Terminal Region of the Heparin-Binding Domain of VEGF165

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid β (Aβ) interactions without affecting other biological activities of VEGF or Aβ [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.

Structural and Dynamic Features of Cold-Shock Proteins of Listeria monocytogenes, a Psychrophilic Bacterium

Cold-shock proteins (Csps), proteins expressed when the ambient temperature drops below the growth-supporting temperature, bind to single-stranded nucleic acids and act as RNA chaperones to regulate translation. Listeria monocytogenes is a psychrophilic food-borne pathogen that is problematic for the food industry. Structures of Csps from psychrophilic bacteria have not yet been studied. Despite dramatic differences in the thermostability of Csps of various thermophilic microorganisms, these proteins share a high degree of primary sequence homology and a high degree of three-dimensional structural similarity. Here, we investigated the structural and dynamic features as well as the thermostability of L. monocytogenes CspA (Lm-CspA). Lm-CspA has a five-stranded β-barrel structure with hydrophobic core packing and two salt bridges. When heptathymidine (dT(7)) binds, values for the heteronuclear nuclear Overhauser effect and order parameters of residues in surface loop regions near nucleic acid binding sites increase dramatically. Moreover, Carr-Purcell-Meiboom-Gill experiments showed that slow motions observed for the nucleic acid binding residues K7, W8, F15, F27, and R56 disappeared in Lm-CspA-dT(7). Lm-CspA is less thermostable than mesophilic and thermophilic Csps, with a lower melting temperature (40 °C). The structural flexibility that accompanies longer surface loops and less hydrophobic core packing and a number of salt bridges and unfavorable electrostatic repulsion are likely key factors in the low thermostability of Lm-CspA. This implies that the large conformational flexibility of psychrophilic Lm-CspA, which more easily accommodates nucleic acids at low temperature, is required for RNA chaperone function under cold-shock conditions and for the cold adaptation of L. monocytogenes.

Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics

Thioredoxins (TRXs) constitute attractive α/β scaffolds for investigating molecular recognition. The interaction between the recombinant fragment spanning the sequence 1-93 of full-length TRX (TRX1-93) and the synthetic peptide comprising residues 94-108 (TRX94-108), plus a C-terminal tyrosine tag (the numbering scheme used in entry pdb 2TRX is used throughout the article, two complementary moieties of E. coli TRX, brings about the consolidation of a native-like complex. Despite its reduced thermodynamic stability, this complex is able to acquire fine structural features remarkably similar to those characteristic of full-length TRX, namely, hydrodynamic behavior, assessed by diffusion-ordered spectroscopy (DOSY)-NMR; the pattern of secondary structure, as revealed by three-bond HNHα coupling constants and secondary shifts for Hα/CO/Cα/Cβ; native-like tertiary structural signatures revealed by near-UV circular dichroism (CD) spectroscopy. The complex exhibits a relaxation behavior compatible with that expected for a native-like structure. However, heteronuclear nuclear Overhauser effect (NOE)s reveal an enhanced dynamics for the complex by comparison with full-length TRX. Furthermore, higher R(2) values for residues 43-50 and 74-89 would likely result from an exchange process modulated by the peptide at the interface region. The slow kinetics of the consolidation reaction was followed by CD and real-time NMR. Equilibrium titration experiments by NMR yield a K(D) value of 1.4 ± 1.0 μM and a second low-affinity (>150 μM) binding event in the vicinity of the active site. Molecular dynamics simulations of both the isolated fragment TRX1-93 and the complex suggest the destabilization of α2 and α3 helical elements and the persistence of β-structure in the absence of TRX94-108. Altogether, structural and dynamic evidence presented herein points to the key role played by the C-terminal helix in establishing the overall fold. This critical switch module endows reduced TRX with the ability to act as a cooperative folding unit.

Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal β-hairpin conformation

Trichomonas vaginalis Myb3 transcription factor (tvMyb3) recognizes the MRE-1 promoter sequence and regulates ap65-1 gene, which encodes a hydrogenosomal malic enzyme that may play a role in the cytoadherence of the parasite. Here, we identified tvMyb353–180 as the essential fragment for DNA recognition and report the crystal structure of tvMyb353–180 bound to MRE-1 DNA. The N-terminal fragment adopts the classical conformation of an Myb DNA-binding domain, with the third helices of R2 and R3 motifs intercalating in the major groove of DNA. The C-terminal extension forms a β-hairpin followed by a flexible tail, which is stabilized by several interactions with the R3 motif and is not observed in other Myb proteins. Interestingly, this unique C-terminal fragment does not stably connect with DNA in the complex structure but is involved in DNA binding, as demonstrated by NMR chemical shift perturbation, 1H-15N heteronuclear-nuclear Overhauser effect and intermolecular paramagnetic relaxation enhancement. Site-directed mutagenesis also revealed that this C-terminal fragment is crucial for DNA binding, especially the residue Arg153 and the fragment K170KRK173. We provide a structural basis for MRE-1 DNA recognition and suggest a possible post-translational regulation of tvMyb3 protein.

Residue-Resolved Stability of Full-Consensus Ankyrin Repeat Proteins Probed by NMR

We investigated the stability determinants and the unfolding characteristics of full-consensus designed ankyrin repeat proteins (DARPins) by NMR. Despite the repeating sequence motifs, the resonances could be fully assigned using 2H, 15N, 13C triple-labeled proteins. To remove further ambiguities, we attached paramagnetic spin labels to either end of these elongated proteins, which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with two and three internal repeats between N- and C-terminal capping repeats (NI 2C, NI 3C) and NI 3C_Mut5, where the C-cap had been reengineered, indicate that the stability of the full-consensus ankyrin repeat proteins is strongly dependent on the coupling between repeats, as the stabilized cap decreases the exchange rate throughout the whole protein. Some amide protons require more than a year to exchange at 37 °C, highlighting the extraordinary stability of the proteins. Denaturant-induced unfolding, followed by deuterium exchange, chemical shift change, and heteronuclear nuclear Overhauser effects, is consistent with an Ising-type description of equilibrium folding for NI 3C_Mut5, while for native-state deuterium exchange, we postulate local fluctuations to dominate exchange as unfolding events are too slow in these very stable proteins. The location of extraordinarily slowly exchanging protons indicates a very stable core structure in the DARPins that combines hydrophobic shielding with favorable electrostatic interactions. These investigations help the understanding of repeat protein architecture and the further design of DARPins for biomedical applications where high stability is required.

Characterization of the Structure and Intermolecular Interactions between the Connexin40 and Connexin43 Carboxyl-terminal and Cytoplasmic Loop Domains

Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the "particle-receptor" model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 15N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36–Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs–ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

Inhibitory Mechanism of Escherichia coli RelE-RelB Toxin-Antitoxin Module Involves a Helix Displacement Near an mRNA Interferase Active Site

In Escherichia coli, RelE toxin participates in growth arrest and cell death by inducing mRNA degradation at the ribosomal A-site under stress conditions. The NMR structures of a mutant of E. coli RelE toxin, RelE(R81A/R83A), with reduced toxicity and its complex with an inhibitory peptide from RelB antitoxin, RelB(C) (Lys(47)-Leu(79)), have been determined. In the free RelE(R81A/R83A) structure, helix alpha4 at the C terminus adopts a closed conformation contacting with the beta-sheet core and adjacent loops. In the RelE(R81A/R83A)-RelB(C) complex, helix alpha3(*) of RelB(C) displaces alpha4 of RelE(R81A/R83A) from the binding site on the beta-sheet core. This helix replacement results in neutralization of a conserved positively charged cluster of RelE by acidic residues from alpha3(*) of RelB. The released helix alpha4 becomes unfolded, adopting an open conformation with increased mobility. The displacement of alpha4 disrupts the geometry of critical residues, including Arg(81) and Tyr(87), in a putative active site of RelE toxin. Our structures indicate that RelB counteracts the toxic activity of RelE by displacing alpha4 helix from the catalytically competent position found in the free RelE structure.

Structures of two Arabidopsis thaliana major latex proteins represent novel helix‐grip folds

Here we report the first structures of two major latex proteins (MLPs) which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (Swiss- Prot/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as foldspace targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17-173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobicmore » binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general.« less

Unique Backbone-Water Interaction Detected in Sphingomyelin Bilayers with 1H/ 31P and 1H/ 13C HETCOR MAS NMR Spectroscopy

Two-dimensional 1H/ 31P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving 31P have not been previously pursued to a great extent in lipid bilayers due to the long 1H- 31P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different 1H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these 1H/ 31P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional 1H/ 13C INEPT HETCOR experiments with NOE mixing support the 1H/ 31P dipolar HETCOR results and confirm the presence of a H 2O environment that has nonvanishing dipolar interactions with the SM backbone.

Crystal Structure of the BARD1 Ankyrin Repeat Domain and Its Functional Consequences

BARD1 is the constitutive nuclear partner to the breast and ovarian cancer-specific tumor suppressor BRCA1. Together, they form a heterodimeric complex responsible for maintaining genomic stability through nuclear functions involving DNA damage signaling and repair, transcriptional regulation, and cell cycle control. We report the 2.0A structure of the BARD1 ankyrin repeat domain. The structure includes four ankyrin repeats with a non-canonical C-terminal capping ankyrin repeat and a well ordered extended loop preceding the first repeat. Conserved surface features show an acidic patch and an acidic pocket along the surface typically used by ankyrin repeat domains for binding cognate proteins. We also demonstrate that two reported mutations, N470S and V507M, in the ankyrin repeat domain do not result in observable structural defects. These results provide a structural basis for exploring the biological function of the ankyrin repeat domain and for modeling BARD1 isoforms.

Structural characterization of partially folded intermediates of apomyoglobin H64F

We present a detailed investigation of unfolded and partially folded states of a mutant apomyoglobin (apoMb) where the distal histidine has been replaced by phenylalanine (H64F). Previous studies have shown that substitution of His64, located in the E helix of the native protein, stabilizes the equilibrium molten globule and native states and leads to an increase in folding rate and a change in the folding pathway. Analysis of changes in chemical shift and in backbone flexibility, detected via [1H]-15N heteronuclear nuclear Overhauser effect measurements, indicates that the phenylalanine substitution has only minor effects on the conformational ensemble in the acid- and urea-unfolded states, but has a substantial effect on the structure, dynamics, and stability of the equilibrium molten globule intermediate formed near pH 4. In H64F apomyoglobin, additional regions of the polypeptide chain are recruited into the compact core of the molten globule. Since the phenylalanine substitution has negligible effect on the unfolded ensemble, its influence on folding rate and stability comes entirely from interactions within the compact folded or partly folded states. Replacement of His64 with Phe leads to favorable hydrophobic packing between the helix E region and the molten globule core and leads to stabilization of helix E secondary structure and overall thermodynamic stabilization of the molten globule. The secondary structure of the equilibrium molten globule parallels that of the burst phase kinetic intermediate; both intermediates contain significant helical structure in regions of the polypeptide that comprise the A, B, E, G, and H helices of the fully folded protein.

Crystallographic and NMR Analyses of UvsW and UvsW.1 from Bacteriophage T4

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3'-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7A and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases.