Preparation and Characterization of a Small Library of Thermally-Labile End-Caps for Variable-Temperature Triggering of Self-Immolative Polymers.

The reaction between furans and maleimides has increasingly become a method of interest as its reversibility makes it a useful tool for applications ranging from self-healing materials, to self-immolative polymers, to hydrogels for cell culture and for the preparation of bone repair. However, most of these applications have relied on simple monosubstituted furans and simple maleimides and have not extensively evaluated the potential thermal variability inherent in the process that is achievable through simple substrate modification. A small library of cycloadducts suitable for the above applications was prepared, and the temperature dependence of the retro-Diels-Alder processes was determined through in situ 1H NMR analyses complemented by computational calculations. The practical range of the reported systems ranges from 40 to >110 °C. The cycloreversion reactions are more complex than would be expected based on simple trends expected based on frontier molecular orbital analyses of the materials.

Conformational Study of N,N'-Diacyl Bispidines and Dioxo Bis-bispidines: Planar Chirality and Molecular Switching.

N,N'-Diacyl bispidines exhibit chirality in the absence of a chiral center and axis. Conformational analysis indicates planar chirality in the molecular structure as a result of open-ended chiral planes, which has been confirmed by X-ray diffraction studies. Substantial chiral-achiral molecular switching is observed in di(haloacetyl) bispidines upon changing the solvent polarity. Tethering the chiral planes with a bispidine linker alters the planar chirality significantly and renders the resulting bis-bispidine macrocycles achiral.

SAGA: rapid automatic mainchain NMR assignment for large proteins.

Here we describe a new algorithm for automatically determining the mainchain sequential assignment of NMR spectra for proteins. Using only the customary triple resonance experiments, assignments can be quickly found for not only small proteins having rather complete data, but also for large proteins, even when only half the residues can be assigned. The result of the calculation is not the single best assignment according to some criterion, but rather a large number of satisfactory assignments that are summarized in such a way as to help the user identify portions of the sequence that are assigned with confidence, vs. other portions where the assignment has some correlated alternatives. Thus very imperfect initial data can be used to suggest future experiments.

Measurement and interpretation of 15N-1H residual dipolar couplings in larger proteins.

A decade ago, Dr. L.E. Kay and co-workers described an ingenious HNCO-based triple-resonance experiment from which several protein backbone RDCs can be measured simultaneously (Yang et al. (1999) [1]). They implemented a J-scaling technique in the (15)N dimension of the 3D experiment to obtain the NH RDCs. We have used this idea to carry out J-scaling in a 2D (15)N-(1)H-TROSY experiment and have found it to be an excellent method to obtain NH RDCs for larger proteins upto 70 kDa, far superior to commonly used HSQC in-phase/anti-phase and HSQC/TROSY comparisons. Here, this method, dubbed "RDC-TROSY" is discussed in detail and the limits of its utility are assessed by simulations. Prominent in the latter analysis is the evaluation of the effect of amide proton flips on the "RDC-TROSY" linewidths. The details of the technical and computational implementations of these methods for the determination of domain orientations in 45-60 kDa Hsp70 chaperone protein constructs are described.

Analysis of the structure of human apo-S100B at low temperature indicates a unimodal conformational distribution is adopted by calcium-free S100 proteins.

S100B is one of the best-characterized members of the calcium-signaling S100 protein family. Most S100 proteins are dimeric, with each monomer containing two EF-hand calcium-binding sites (EF1, EF2). S100B and other S100 proteins respond to calcium increases in the cell by coordinating calcium and undergoing a conformational change that allows them to interact with a variety of cellular targets. Although several three dimensional structures of S100 proteins are available in the calcium-free (apo-) state it has been observed that these structures appear to adopt a wide range of conformations in the EF2 site with respect to the positioning of helix III, the helix that undergoes the most dramatic calcium-induced conformational change. In this work, we have determined the structure of human apo-S100B at 10 degrees C to examine whether temperature might be responsible for these structural differences. Further, we have used this data, and other available apo-S100 structures, to show that despite the range of interhelical angles adopted in the apo-S100 structures, normal Gaussian distributions about the mean angles found in the structure of human apo-S100B are observed. This finding, only obvious from the analysis of all available apo-S100 proteins, provides direct structural evidence that helix III is a loosely packed helix. This is likely a necessary functional property of the S100 proteins that facilitates the calcium-induced conformational change of helix III. In contrast, the calcium-bound structures of the S100 proteins show significantly smaller variability in the interhelical angles. This shows that calcium binding to the S100 proteins causes not only a conformational change but results in a tighter distribution of helices within the EF2 calcium binding site required for target protein interactions.

Solution structure of the Escherichia coli protein ydhR: a putative mono-oxygenase.

YdhR is a 101-residue conserved protein from Escherichia coli. Sequence searches reveal that the protein has >50% identity to proteins found in a variety of other bacterial genomes. Using size exclusion chromatography and fluorescence spectroscopy, we determined that ydhR exists in a dimeric state with a dissociation constant of approximately 40 nM. The three-dimensional structure of dimeric ydhR was determined using NMR spectroscopy. A total of 3400 unambiguous NOEs, both manually and automatically assigned, were used for the structure calculation that was refined using an explicit hydration shell. A family of 20 structures was obtained with a backbone RMSD of 0.48 A for elements of secondary structure. The structure reveals a dimeric alpha,beta fold characteristic of the alpha+beta barrel superfamily of proteins. Bioinformatic approaches were used to show that ydhR likely belongs to a recently identified group of mono-oxygenase proteins that includes ActVA-Orf6 and YgiN and are involved in the oxygenation of polyaromatic ring compounds.

NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperone.

Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.

NMR study of nucleotide-induced changes in the nucleotide binding domain of Thermus thermophilus Hsp70 chaperone DnaK: implications for the allosteric mechanism.

We present an NMR investigation of the nucleotide-dependent conformational properties of a 44-kDa nucleotide binding domain (NBD) of an Hsp70 protein. Conformational changes driven by ATP binding and hydrolysis in the N-terminal NBD are believed to allosterically regulate substrate affinity in the C-terminal substrate binding domain. Several crystal structures of Hsc70 NBDs in different nucleotide states have, however, not shown significant structural differences. We have previously reported the NMR assignments of the backbone resonances of the NBD of the bacterial Hsp70 homologue Thermus thermophilus DnaK in the ADP-bound state. In this study we show, by assigning the NBD with the ATP/transition state analogue, ADP.AlFx, bound, that it closely mimics the ATP-bound state. Chemical shift difference mapping of the two nucleotide states identified differences in a cluster of residues at the interface between subdomains 1A and 1B. Further analysis of the spectra revealed that the ATP state exhibited a single conformation, whereas the ADP state was in slow conformational exchange between a form similar to the ATP state and another state unique to the ADP-bound form. A model is proposed of the allosteric mechanism based on the nucleotide state altering the balance of a dynamic equilibrium between the open and closed states. The observed chemical shift perturbations were concentrated in an area close to a previously described J-domain binding channel, confirming the importance of that region in the allosteric mechanism.

The lateral diffusion of selectively aggregated peptides in giant unilamellar vesicles.

We have systematically investigated the effect of aggregation of a transmembrane peptide on its diffusion in dimyristoylphosphatidylcholine and in palmitoyloleoylphosphatidylcholine model membranes. The hydrophobic segment of the b subunit from E. coli F(1)F(0)-ATP synthase was modified with a histidine tag at the carbonyl terminus and was aggregated selectively by using a series of multivalent, dendritic chelating agents with nitrilotriacetic acid functional groups. Peptide complexes ranging from monomers to hexamers were formed and studied in giant unilamellar vesicles. The rate of diffusion for the transmembrane peptide complexes were found to depend on the size of the complex. The results agree with predictions from the free area model for monomers and dimers, and the hydrodynamic continuum model for tetramers, pentamers, and hexamers. Comparisons with diffusion of lipids confirm that the diffusion of a transmembrane peptide is enhanced by coupling of density fluctuations between the two monolayers.

Folding and stability of the b subunit of the F(1)F(0) ATP synthase.

The F(1)F(0) ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F(0)) and soluble (F(1)) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F(1)F(0) complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N- and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with T(m) values between 30 and 40 degrees C. CD spectra for the region comprising residues 53-122 (b(53-122)) showed theta;(222)/theta;(208) = 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b(53-122) (DeltaG, DeltaH and DeltaC(p)) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.