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The reflective (RC) and anti-reflective coatings (ARC) on freestanding GaN were fabricated using a stack of TiO2/SiO2 multi-layers, which changes the reflectance at 532 nm from 17.0% to 2.5% and 99.6%, respectively, and proves the effectiveness of RC and ARC. The reflection spectra of annealed RC and ARC both show a blueshift of about 52 nm after high-temperature annealing, because of the phase transition of TiO2 from amorphism to anatase at high temperatures. In order to improve the thermal stability of RC and ARC, we could increase the thickness of TiO2 and SiO2 proportionally during the coating process.

The folding of predominantly β-sheet proteins is complicated by the presence of a large number of non-local interactions in their native states, which increase the ruggedness of their folding energy landscapes. However, forming non-local contacts early in folding or even in the unfolded state can smooth the energy landscape and facilitate productive folding. We report that several sequence regions of a β-barrel protein, cellular retinoic acid-binding protein 1 (CRABP1), populate native-like secondary structure to a significant extent in the denatured state in 8 M urea. In addition, we provide evidence for both local and non-local interactions in the denatured state of CRABP1. NMR chemical shift perturbations (CSPs) under denaturing conditions upon substitution of single residues by mutation support the presence of several non-local interactions in topologically key sites, arguing that the denatured state is conformationally restricted and contains topological information for the native fold. Among the most striking non-local interactions are those between the N- and C-terminal regions, which are involved in closure of the native β-barrel. In addition, CSPs support the presence of two features in the denatured state: a major hydrophobic cluster involving residues from various parts of the sequence and a native-like interaction similar to one identified in previous studies as forming early in folding (Budyak et al., Structure 21, 476 [2013]). Taken together, our data support a model in which transient structures involving nonlocal interactions prime early folding interactions in CRABP1, determine its barrel topology, and may protect this predominantly β-sheet protein against aggregation.

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle.

The Hsp70 family of chaperones works with its co-chaperones, the nucleotide exchange factors and J-domain proteins, to facilitate a multitude of cellular functions. Central players in protein homeostasis, these jacks-of-many-trades are utilized in a variety of ways because of their ability to bind with selective promiscuity to regions of their client proteins that are exposed when the client is unfolded, either fully or partially, or visits a conformational state that exposes the binding region in a regulated manner. The key to Hsp70 functions is that their substrate binding is transient and allosterically cycles in a nucleotide-dependent fashion between high- and low-affinity states. In the past few years, structural insights into the molecular mechanism of this allosterically regulated binding have emerged and provided deep insight into the deceptively simple Hsp70 molecular machine that is so widely harnessed by nature for diverse cellular functions. In this review, these structural insights are discussed to give a picture of the current understanding of how Hsp70 chaperones work.

The 70-kDa heat shock proteins (Hsp70s) are molecular chaperones that perform a wide range of critical cellular functions. They assist in the folding of newly synthesized proteins, facilitate assembly of specific protein complexes, shepherd proteins across membranes, and prevent protein misfolding and aggregation. Hsp70s perform these functions by a conserved mechanism that relies on allosteric cycles of nucleotide-modulated binding and release of client proteins. Current models for Hsp70 allostery have come from extensive study of the bacterial Hsp70, DnaK. Extending our understanding to eukaryotic Hsp70s is extremely important not only in providing a likely common mechanistic framework but also because of their central roles in cellular physiology. In this study, we examined the allosteric behaviors of the eukaryotic cytoplasmic Hsp70s, HspA1 and Hsc70, and found significant differences from that of DnaK. We found that HspA1 and Hsc70 favor a state in which the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) are intimately docked significantly more as compared to DnaK. Past work established that the NBD-SBD interface and the helical lid-β-SBD interface govern the allosteric landscape of DnaK. Here, we identified sites on these interfaces that differ between eukaryotic cytoplasmic Hsp70s and DnaK. Our mutational analysis has revealed key evolutionary variations that account for the population shifts between the docked and undocked conformations. These results underline the tunability of Hsp70 functions by modulation of allosteric interfaces through evolutionary diversification and also suggest sites where the binding of small-molecule modulators could influence Hsp70 function.

The folding of predominantly β‐sheet proteins is complicated by the presence of a large number of non‐local interactions in their native states, which increase the ruggedness of their folding energy landscapes. However, forming non‐local contacts early in folding or even in the unfolded state can smooth the energy landscape and facilitate productive folding. We report that several sequence regions of a β‐barrel protein, cellular retinoic acid‐binding protein 1 (CRABP1), populate native‐like secondary structure to a significant extent in the denatured state in 8 M urea. In addition, we provide evidence for both local and non‐local interactions in the denatured state of CRABP1. NMR chemical shift perturbations (CSPs) under denaturing conditions upon substitution of single residues by mutation support the presence of several non‐local interactions in topologically key sites, arguing that the denatured state is conformationally restricted and contains topological information for the native fold. Among the most striking non‐local interactions are those between the N‐ and C‐terminal regions, which are involved in closure of the native β‐barrel. In addition, CSPs support the presence of two features in the denatured state: a major hydrophobic cluster involving residues from various parts of the sequence and a native‐like interaction similar to one identified in previous studies as forming early in folding (Budyak et al., Structure 21, 476 [2013]). Taken together, our data support a model in which transient structures involving nonlocal interactions prime early folding interactions in CRABP1, determine its barrel topology, and may protect this predominantly β‐sheet protein against aggregation.

Hsp70 molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, and more specialized roles such as disassembly of particular protein complexes. It is a fascinating question to ask how the mechanism of these deceptively simple molecular machines is matched to their roles in these wide-ranging processes. The key is a combination of the nature of the recognition and binding of Hsp70 substrates and the impact of Hsp70 action on their substrates. In many cases, the binding, which relies on interaction with an extended, accessible short hydrophobic sequence, favors more unfolded states of client proteins. The ATP-mediated dissociation of the substrate thus releases it in a relatively less folded state for downstream folding, membrane translocation, or hand-off to another chaperone. There are cases, such as regulation of the heat shock response or disassembly of clathrin-coats, however, where binding of a short hydrophobic sequence selects conformational states of clients to favor their productive participation in a subsequent step. This Perspective discusses current understanding of how Hsp70 molecular chaperones recognize and act on their substrates and the relationships between these fundamental processes and the functional roles played by these molecular machines.

The plasminogen activator (PA) in snake venom, a serine protease, can convert plasminogen to active plasmin, indirectly causing the degradation of fibrin. It is difficult to purify sufficient snake venom PA (SV-PA) for clinical applications due to the low SV-PA content in venom. The gene encoding PA was obtained from the venom gland of Gloydius brevicaudus using RT-PCR with primers designed according to the N-terminal amino acids of G. brevicaudus venom PA (GBV-PA), was cloned into the prokaryotic expression vector pET-42a, and recombinant GBV-PA (rGBV-PA) was expressed via Isopropyl-β-d-1-Thiogalactopyranoside (IPTG) induction. Like human tissue PA, the purified renatured rGBV-PA could significantly reduce the rabbit plasma euglobulin lysis time (ELT) and prevent thrombus formation in the inferior vena cava of rats. Within the dosage range, the dosage and effects were positively correlated.

The denatured state ensemble (DSE) represents the starting state for protein folding and the reference state for protein stability studies. Residual structure in the DSE influences the kinetics of protein folding, the propensity to aggregate, and protein stability. The DSE that is most relevant for folding is the ensemble populated under native conditions, but the stability of proteins and the cooperativity of their folding normally prevent direct characterization of this ensemble. Indirect experiments have been used to infer residual structure in the DSE under nondenaturing conditions, but direct characterization is rare. The N-terminal domain of ribosomal protein L9 (NTL9) is a small mixed α-β domain that folds cooperatively on the millisecond time scale. A destabilized double mutant of NTL9, V3A/I4A-NTL9, populates the DSE in the absence of denaturant and is in slow exchange with the native state on the nuclear magnetic resonance time scale. The DSE populated in buffer was compared to the urea-induced DSE. Analysis of (1)H and (13)C chemical shifts reveals residual secondary structure in the DSE in buffer, which is stabilized by both local and long-range interactions. (15)N R2 relaxation rates deviate from random coil models, suggesting hydrophobic clustering in the DSE. Paramagnetic relaxation enhancement experiments show that there are transient long-range contacts in the DSE in buffer. In contrast, the urea-induced DSE has significantly less residual secondary structure and markedly fewer long-range contacts; however, the urea-induced DSE deviates from a random coil.

The pH dependence of protein stability is defined by the difference in the number of protons bound to the folded state and to the denatured state ensemble (DSE) as a function of pH. In many cases, the protonation behavior can be described as the sum of a set of independently titrating residues; in this case, the pH dependence of stability reflects differences in folded and DSE pKa's. pH dependent stability studies have shown that there are energetically important interactions involving charged residues in the DSE of the N-terminal domain of L9 (NTL9), which affect significantly the stability of the protein. The DSE of wild type NTL9 cannot be directly characterized under native conditions because of its high stability. A destabilized double mutant of NTL9, V3AI4A, significantly populates the folded state and the DSE in the absence of denaturant. The two states are in slow exchange on the nuclear magnetic resonance time scale, and diffusion measurements indicate that the DSE is compact. The DSE pKa's of all of the acidic residues were directly determined. The DSE pKa of Asp8 and Asp23 are depressed relative to model compounds values. Use of the mutant DSE pKa's together with known native state pKa's leads to a significantly improved agreement between the measured pH dependent stability and that predicted by the Tanford-Wyman linkage relationship. An analysis of the literature suggests that DSE interactions involving charged residues are relatively common and should be considered in discussions of protein stability. Proteins 2011; © 2011 Wiley-Liss, Inc.

The villin headpiece subdomain, HP36, is the smallest naturally occurring protein that folds cooperatively. Its small size, rapid folding, and simple three-helix topology have made it an extremely popular system for computational studies of protein folding. The role of unfolded state structure in rapid folding is an area of active investigation, but relatively little is known about the properties of unfolded states under native conditions. A peptide fragment, HP21, which contains the first and second helices of HP36 has been shown to be a good model for structure in the unfolded state of the intact domain but a detailed description of the conformational propensities of HP21 is lacking and the balance between native and nonnative interactions is not known. A series of three-dimensional NMR experiments were performed on 13C, 15N-labeled HP21 to investigate in detail its conformational propensities. Analysis of 13Cα, 13Cβ, 13CO chemical shifts, Δδ13Cα − Δδ13Cβ secondary shifts, the secondary structure propensity scores, NOEs, 15N R2 values and comparison of experimental chemical shifts with those of HP36 and with chemical shifts calculated using the SHIFTS and SHIFTX programs all indicate that there is significant native like structure in the HP21 ensemble, and thus by implication in the unfolded state of HP36.

Assembly of bidentate ligand 1-(1-imidazolyl)-4-(imidazol-1-ylmethyl)benzene (IIMB) with varied metal salts of ZnII, CdII and PbII provide three new complexes, [Zn(IIMB)2](ClO4)2·2H2O (1), [Cd(IIMB)2(SCN)2] (2) and [Pb(IIMB)2(SCN)](SCN) (3). Single crystal X-ray diffraction studies revealed that complexes 1 and 2 display a similar one-dimensional double stranded chain structure, while complex 3 is a slight distorted rhombohedral grid network with (4,4) topology. The results indicate that the coordination geometry of the metal ion and the counter anion have great impact on the structure of the complexes. In addition, the photoluminescence properties of ligand IIMB and complexes 1–3 were studied in the solid state at room temperature.

Seven new metal−organic frameworks (MOFs) [Cu(bib)2(H2O)2](Cl3CCOO)2·2MeOH·2H2O (1), [Cu(bib)2]Cl2·H2O (2), [Cu(bib)2(SO4)(H2O)]·4H2O (3), [Cu(bib)(SO4)(H2O)2] (4), [Cu(bib)(N3)(OAc)]·DMF (5) (OAc = acetate), [Cu(bib)(ox)]·MeOH·2H2O (6) (ox = oxalate), and [Cu(bib)(mal)]·MeOH (7) (mal = malonate) were obtained by reactions of flexible bidentate ligand 1-bromo-3,5-bis(imidazol-1-ylmethyl)benzene (bib) with corresponding copper(II) salts. The structures of these MOFs were established by single-crystal X-ray diffraction analysis. Complexes 1 and 2 have similar one-dimensional (1D) hinged chain structure. Complex 3 has a 2D network structure with (4,4) topology. Complex 4 has M2L2 metallocyclic ring structure and the rings are further linked by the sulfate anions to form a 1D tube-like chain. Complex 5 is also a dinuclear M2L2 metallocyclic ring, which is further connected by Br···Br interactions to form 1D chain. While in the cases of complexes 6 and 7, both have 2D network structure in which the oxalate and...

Three novel copper complexes, [CuII(titmb)(N3)2]·0.5H2O (1), [CuI3(titmb)2](ClO4)3 (2), and [CuII(titmb)(SO4)]·2.2H2O (3), were obtained by reactions of flexible tripodal ligand 1,3,5-tris(imidazol-1-ylmethyl)-2,4,6-trimethylbenzene (titmb) with the corresponding copper(II) salts using hydrothermal method and their structures were determined by X-ray crystallography. Complex 1 exhibits a one-dimensional (1D) tube-like chain structure, while the 2 possesses a M3L2 molecular cage-like structure, in which the copper atom has a valence of +1, indicating that the CuII is reduced to CuI under the hydrothermal condition. Complex 3 has a polycatenated network structure with inclined interpenetration of (6,3) networks. Ligand titmb adopts a cis,cis,cis-conformation in all of the three complexes. The results indicate that the anions play important roles in the structure of the complexes. In addition, complex 2 was further investigated by electrospray massspectrometry.

Three metal-organic frameworks (MOFs) [La(bta)(H2O)]n (1), [La(bta)(H2O)2·H2O]n (2) and [Er(bta)(H2O)2·H2O]n (3) with different structures were synthesized by reactions of 1,3,5-benzenetriacetic acid (H3bta) with the corresponding lanthanide salts under different conditions. X-ray diffraction analyses reveal that complexes 1 and 2 have the same metal atom and ligand but different structures. In 1, each bta3 − adopts cis, cis, cis conformation and acts as a μ6-bridge linking six lanthanum atoms to form a 2D framework; while in 2, the bta3 − has cis, trans, trans conformation and serves as a μ5-bridging ligand, which results in a 3D channel-like structure. In the case of 3, each bta3 − also adopts cis, trans, trans conformation but acts as a μ4-bridge linking four erbium atoms to form a 3D channel-like framework. The results imply that the reaction conditions have great impact on the structure of MOFs, and the flexible triacid ligand H3bta is versatile and can adopt different conformations in the formati...

A new complex [Pr(bib)2(NO3)3] (1) was synthesized by reaction of bidentate imidazole-containing ligand 1-bromo-3,5-bis(imidazol-1-ylmethyl)benzene (bib) with Pr(NO3)·6H2O and characterized by X-ray crystallography. Complex 1 has a two-dimensional herringbone-like structure with the ligand bib serving as a bridging ligand using its two imidazolyl nitrogen atoms. Ligand bib adopts cis and trans two different conformations, and the Pr(III) atoms are bridged by bib in two different ways. Thermogravimetric analysis for complex 1 was carried out and the result shows that the complex is stable up to 180 °C. Variable-temperature magnetic susceptibility of complex 1 was measured between 1.8 and 300 K and the result shows that the χMT value decreases continuously over the whole temperature range. Copyright © 2005 John Wiley & Sons, Ltd.

Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pKa measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.

The sizes of unfolded proteins under highly denaturing conditions scale as N 0.59 with chain length. This suggests that denaturing conditions mimic good solvents, whereby the preference for favorable chain–solvent interactions causes intrachain interactions to be repulsive, on average. Beyond this generic inference, the broader implications of N 0.59 scaling for quantitative descriptions of denatured state ensembles (DSEs) remain unresolved. Of particular interest is the degree to which N 0.59 scaling can simultaneously accommodate intrachain attractions and detectable long-range contacts. Here we present data showing that the DSE of the N-terminal domain of the L9 (NTL9) ribosomal protein in 8.3 M urea lacks detectable secondary structure and forms expanded conformations in accord with the expected N 0.59 scaling behavior. Paramagnetic relaxation enhancements, however, indicate the presence of detectable long-range contacts in the denatured-state ensemble of NTL9. To explain these observations we used atomistic thermal unfolding simulations to identify ensembles whose properties are consistent with all of the experimental observations, thus serving as useful proxies for the DSE of NTL9 in 8.3 M urea. Analysis of these ensembles shows that residual attractions are present under mimics of good solvent conditions, and for NTL9 they result from low-likelihood, medium/long-range contacts between hydrophobic residues. Our analysis provides a quantitative framework for the simultaneous observation of N 0.59 scaling and low-likelihood long-range contacts for the DSE of NTL9. We propose that such low-likelihood intramolecular hydrophobic clusters might be a generic feature of DSEs that play a gatekeeping role to protect against aggregation during protein folding.

According to the geometric imaging characteristics of airborne Synthetic Aperture Radar (SAR) system, the methods of extracting three dimension information for some typical ground objects with height have been described in this paper, including image preprocessing, height information extracting of buildings and trees, the final visualization and so on. And, the test result provided a practical reference for the application of urban and town area with high-resolution airborne SAR images.

Interactions in protein denatured state ensembles (DSE) influence protein stability and folding kinetics. Characterization of DSE is difficult because of the underlying conformational heterogeneity. Recent paramagnetic relaxation enhancement (PRE) measurements show evidence for persistent non-local interactions in the N-terminal domain of ribosomal protein L9 (NTL9) under strongly denaturing conditions. Surprisingly, these contacts between residues distal in sequence prevail despite the lack of detectable secondary structure and evidence that the protein samples significantly expanded conformations. We present results from computational thermal unfolding studies on the NTL9 protein to provide an explanation for the observed long-range contacts. The simulations are based on the ABSINTH implicit solvation model and utilize efficient Monte Carlo methods for conformational sampling. We identify a temperature range that yields ensembles consistent with the experimental data as inferred by direct, quantitative comparison of experimental observables from simulation results to experimental data. We refer to these ensembles as TD-ensembles which were generated without any a priori knowledge of the experimental data and analyze these ensembles to provide a quantitative interpretation of experimental results. Persistent non-local contacts are made throughout the NTL9 DSE without appreciable secondary structure; the most significant being relatively low-likelihood hydrophobic clusters and contacts between the N-terminal and central portion of NTL9. Our results explain how the average dimensions of denatured proteins conform to the statistics of self-avoiding random walks despite sampling conformations with low likelihoods of long-range contacts.

The nature of the denatured state ensemble is controversial owing in large part to the difficulty of characterizing the structure and energetics of denatured state interactions. Denatured states can be populated under a variety of extreme conditions but the state which is most relevant for protein folding and engineering is the denatured state ensemble which is populated in the absence of denaturant under native conditions. Unfortunately this state is usually experimentally inaccessible. We reported detailed characterized of the denatured state populated under native conditions for two α-β proteins, the N-terminal domain of the ribosomal protein L9 (NTL9) and the C-terminal domain of the same protein (CTL9) , as well as for a rapid folding all helical structure the villin headpiece helical subdomain, HP-36. Conditions have been found where the native and denatured states of CTL9 are both populated in the absence of denaturant and 1H, 15N and 13C NMR was used to define the conformation propensities of the denatured state. For NTL9 the thermodynamic linkage between proton binding and protein stability was used to characterize denatured state electrostatic interactions. Peptide models were exploited to characterize the denatured state of HP-36. In all three cases, the denatured states contains significant structure. The impact of this preformed structure on the kinetics and mechanism of protein folding is discussed.