Search

Since the emergence of organometal halide perovskite (OMP) solar cells, there has been growing interest in the benefits of incorporating polymer additives into the perovskite precursor, in terms of both photovoltaic device performance and perovskite stability. In addition, there is interest in the self-healing properties of polymer-incorporated OMPs, but the mechanisms behind these enhanced characteristics are still not fully understood. Here, we study the role of poly(2-hydroxyethyl methacrylate) (pHEMA) in improving the stability of methylammonium lead iodide (MAPI, CH 3 NH 3 PbI 3 ) and determine a mechanism for the self-healing of the perovskite-polymer composite following exposure to atmospheres of differing relative humidity, using photoelectron spectroscopy. Varying concentrations of pHEMA (0-10 wt %) are incorporated into a PbI 2 precursor solution during the conventional two-step fabrication method for producing MAPI. It is shown that the introduction of pHEMA results in high-quality MAPI films with increased grain size and reduced PbI 2 concentration compared with pure MAPI films. Devices based on pHEMA-MAPI composites exhibit an improved photoelectric conversion efficiency of 17.8%, compared with 16.5% for a pure MAPI device. pHEMA-incorporated devices are found to retain 95.4% of the best efficiency after ageing for 1500 h in 35% RH, compared with 68.5% achieved from the pure MAPI device. The thermal and moisture tolerance of the resulting films is investigated using X-ray diffraction, in situ X-ray photoelectron spectroscopy (XPS), and hard XPS (HAXPES). It is found that exposing the pHEMA films to cycles of 70 and 20% relative humidity leads to a reversible degradation, via a self-healing process. Angle-resolved HAXPES depth-profiling using a non-destructive Ga Kα source shows that pHEMA is predominantly present at the surface with an effective thickness of ca. 3 nm. It is shown using XPS that this effective thickness reduces with increasing temperature. It is found that N is trapped in this surface layer of pHEMA, suggesting that N-containing moieties, produced during reaction with water at high humidity, are trapped in the pHEMA film and can be reincorporated into the perovskite when the humidity is reduced. XPS results also show that the inclusion of pHEMA enhances the thermal stability of MAPI under both UHV and 9 mbar water vapor pressure.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

AFM-IR combines the chemical sensitivity of infrared spectroscopy with the lateral resolution of scanning probe microscopy, allowing nanoscale chemical analysis of almost any organic material under ambient conditions. As a result, this versatile technique is rapidly gaining popularity among materials scientists. Here, we report a previously overlooked source of data and artifacts in AFM-IR analysis; reflection from the buried interface. Periodic arrays of gold on glass are used to show that the overall signal in AFM-IR is affected by the wavelength-dependent reflectivity and thermal response of the underlying substrate. Excitingly, this demonstrates that remote analysis of heterogeneities at the buried interface is possible alongside that of an overlying organic film. On the other hand, AFM-IR users should carefully consider the composition and topography of underlying substrates when interpreting nanoscale infrared data. The common practice of generating ratio images, or indeed the normalization of AFM-IR spectra, should be approached with caution in the presence of substrate heterogeneity or variable sample thickness.

In this report, we describe a new photoredox catalyzed 1,4-conjugate addition of N -substituted acetic acids to electron-deficient olefins via decarboxylative C-C bond formation. This C-C bond formation occurred under mild conditions enabled by visible light irradiation. This transformation facilitated the synthesis of biologically relevant N -substituted heterocyclic structural motifs not readily accessible by other methods. The C-C bond formation protocol was applied to weakly nucleophilic heterocycles such as indoles, indazoles, imidazoles, and cyclic amides to form functionalized drug-like small molecule.

The diffusion of rhodamine-labeled poly(ethylene glycol) (r-PEG) within surface-grafted poly(ethylene glycol) (s-PEG) layers in aqueous solution at 18 °C was measured by fluorescence correlation spectroscopy. The diffusion coefficient of r-PEG within s-PEG was controlled by the grafting density, σ, and scaled as σ -1.42±0.09 . It is proposed that a characteristic blob size associated with the grafted (brush) layer defines the region through which the r-PEG diffusion occurs. The diffusion coefficients for r-PEG in semidilute solution were found to be similar to those in the brushes., Competing Interests: The authors declare no competing financial interest.

Using an electrostatic-based super inkjet printer we report the high-resolution deposition of polyelectrolyte macroinitiators and subsequent polymer brush growth using SI-ARGET-ATRP. We go on to demonstrate for the first time a submicron patterning phenomenon through the addition of either a like charged polyelectrolyte homopolymer or through careful control of ionic strength. As a result patterning of polymer brushes down to ca. 300 nm is reported. We present a possible mechanistic model and consider how this may be applied to other polyelectrolyte-based systems as a general method for submicron patterning.

The anion-specific solvation and conformational behavior of weakly basic poly(2-dimethylamino)ethyl methacrylate (poly(DMA)), poly(2-diethylamino)ethyl methacrylate (poly(DEA)), and poly(2-diisopropylamino)ethyl methacrylate (poly(DPA)) brushes, with correspondingly increasing inherent hydrophobicity, have been investigated using in situ ellipsometric and quartz crystal microbalance with dissipation (QCM-D) measurements. In the osmotic brush regime, as the initial low concentration of salt is increased, the brushes osmotically swell by the uptake of solvent as they become charged and the attractive hydrophobic inter- and intrachain interactions are overcome. With increased ionic strength, the brushes move into the salted brush regime where they desolvate and collapse as their electrostatic charge is screened. Here, as the brushes collapse, they transition to more uniform and rigid conformations, which dissipate less energy, than similarly solvated brushes at lower ionic strength. Significantly, in these distinct regimes brush behavior is not only ionic strength dependent but is also influenced by the nature of the added salt based on its position in the well-known Hofmeister or lyotropic series, with potassium acetate, nitrate, and thiocyanate investigated. The strongly kosmotropic acetate anions display low affinity for the hydrophobic polymers, and largely unscreened electrosteric repulsions allow the brushes to remain highly solvated at higher acetate concentrations. The mildly chaotropic nitrate and strongly chaotropic thiocyanate anions exhibit a polymer hydrophobicity-dependent affinity for the brushes. Increasing thiocyanate concentration causes the brushes to collapse at lower ionic strength than for the other two anions. This study of weak polybasic brushes demonstrates the importance of all ion, solvent, and polymer interactions.

The swelling behavior of poly(2-(diethylamino)ethyl methacrylate) (PDEA) brushes in response to changes in solution pH and ionic strength has been investigated. The brushes were synthesized by ARGET ATRP methodology at the silica-aqueous solution interface via two different surface-bound initiator approaches: electrostatically adsorbed cationic macroinitiator and covalently anchored silane-based ATRP initiator moieties. The pH-response of these brushes is studied as a function of the solvated brush thickness in a constant flow regime that elucidates the intrinsic behavior of polymer brushes. In situ ellipsometry equilibrium measurements show the pH-induced brush swelling and collapse transitions are hysteretic in nature. Furthermore, high temporal resolution kinetic studies demonstrate that protonation and solvent ingress during swelling occur much faster than the brush charge neutralization and solvent expulsion during collapse. This hysteresis is attributed to the formation of a dense outer region or skin during collapse that retards solvent egress. Moreover, at a constant pH below its pKa, the PDEA brush exhibited a critical conformational change in the range 0.5-1 mM electrolyte, a range much narrower than predicted by the theory of the osmotic brush regime. This behavior is attributed to the hydrophobicity of the collapsed brush. The swelling and collapse kinetics for this salt-induced transition are nearly identical. This is in contrast to the asymmetry in the rate of the pH-induced response, suggesting an alternative mechanism for the two processes dependent on the nature of the environmental trigger.

Coatings consisting of polymer brushes are an effective way to modify solid interfaces. Polymer brush-modified hybrid particles have been prepared by surface-initiated activators regenerated by electron transfer atom transfer radical polymerization (SI-ARGET ATRP) of 2-(diethylamino)ethyl methacrylate (DEA) on silica particles. We have optimized the synthesis with respect to changing the reducing agent, temperature, and reaction solvent from an aqueous ethanol mixture to an aqueous methanol mixture. Our flexible electrostatically adsorbed macroinitiator approach allows for the modification of a variety of surfaces. Polybasic brushes have been grown on silica particles of different sizes, from 120 to 840 nm in diameter, as well as on wafers, and a comparison of the products has allowed the effect of surface curvature to be elucidated. An examination of the thickness of the dry brush and the aqueous hydrodynamic brush at both pH 7 and at 4 demonstrated that growth increased substantially with substrate curvature for particles with a diameter below 450 nm. This is attributed to the increasing separation between active chain ends, reducing the rate of termination. This is believed to be the first time that this effect has been demonstrated experimentally. Furthermore, we have seen that polymer brush growth on planar wafers was significantly reduced when the reaction mixture was stirred.

Brush-modified silica hybrids have been synthesized by growing poly(2-(diethylamino)ethyl methacrylate) (poly(DEA)) brushes on 120 nm diameter silica particles by surface-initiated activators regenerated by electron transfer atom transfer radical polymerization (SI-ARGET ATRP). This is the first report of using SI-ARGET ATRP to synthesize poly(DEA) brushes. The kinetics of poly(DEA) brush growth in 4:1 v/v ethanol/water was monitored. The hydrodynamic diameter of the resulting brush-modified particles was dependent on the solution pH due to the weak polybasic nature of the brushes. Below the p K a of poly(DEA), the hydrodynamic diameter of the brush-modified particles increased with decreasing pH as a consequence of brush protonation, rearrangement and solvent uptake. This pH-response of the brushes was reversible and the hybrid particles exhibited significant hydrodynamic volume changes of up to 200% when the solution pH was cycled from pH 7 to pH 4.

We have used neutron reflectometry to investigate the behavior of a strong polyelectrolyte brush on a sapphire substrate, grown by atom-transfer radical polymerization (ATRP) from a silane-anchored initiator layer. The initiator layer was deposited from vapor, following treatment of the substrate with an Ar/H(2)O plasma to improve surface reactivity. The deposition process was characterized using X-ray reflectometry, indicating the formation of a complete, cross-linked layer. The brush was grown from the monomer [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), which carries a strong positive charge. The neutron reflectivity profile of the swollen brush in pure water (D(2)O) showed that it adopted a two-region structure, consisting of a dense surface region ∼100 Å thick, in combination with a diffuse brush region extending to around 1000 Å from the surface. The existence of the diffuse brush region may be attributed to electrostatic repulsion from the positively charged surface region, while the surface region itself most probably forms due to polyelectrolyte adsorption to the hydrophobic initiator layer. The importance of electrostatic interactions in maintaining the brush region is confirmed by measurements at high (1 M) added 1:1 electrolyte, which show a substantial transfer of polymer from the brush to the surface region, together with a strong reduction in brush height. On addition of 10(-4) M oppositely charged surfactant (sodium dodecyl sulfate), the brush undergoes a dramatic collapse, forming a single dense layer about 200 Å in thickness, which may be attributed to the neutralization of the monomers by adsorbed dodecyl sulfate ions in combination with hydrophobic interactions between these dodecyl chains. Subsequent increases in surfactant concentration result in slow increases in brush height, which may be caused by stiffening of the polyelectrolyte chains due to further dodecyl sulfate adsorption.

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes grown by surface-initiated polymerization from a polyanionic macroinitiator adsorbed at the sapphire-water interface have been used as a substrate to study the interaction between the weak polyelectrolyte PDMAEMA and the oppositely charged surfactant sodium dodecyl sulfate (SDS) with neutron reflectivity. At pH 3, multilayered structures are formed in which the interlayer separation (∼40 Å) is comparable to the dimensions of a SDS bilayer or micelle. The number of repeating layers that form depends on brush thickness, ranging from three layers in a relatively thin brush (5 nm dry thickness) to 15 layers in a relatively thick brush (17 nm dry thickness). In the 5 nm brush, addition of 0.01 mM SDS leads to brush deswelling, and the distinct layered structure only forms when the SDS concentration reaches 1 mM, with the brush reswelling slightly at 5 mM SDS. In the thicker (11 and 17 nm) brushes, distinct layered structures form at 0.1 mM SDS, in which the molar SDS/DMAEMA ratio is greater than unity. Exposing the 17 nm brush/SDS complex to 1 M NaNO(3) results in the complete removal of the surfactant and recovery of the bare brush structure. At pH 9, there is significant surfactant uptake by the brush, but no multilayer structures are formed. The brush presents a high concentration of DMAEMA segments that are localized to within 500-1000 Å of the sapphire interface. At pH 9 the high local concentration of hydrocarbon segments in the brush screens the hydrophobic tails of the surfactants from the unfavorable interaction with water, leading to significant surfactant uptake by the brush. At pH 3 the high local concentration of charges inside the brush additionally screens the repulsive interactions between the surfactant headgroups, making surfactant uptake even more favorable, leading to the formation of multilayered surfactant aggregates confined within the brush., (© 2011 American Chemical Society)

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes have been grown by surface-initiated atom transfer radical polymerization (SI-ATRP) from a polyanionic macroinitiator adsorbed at the sapphire-water interface, and neutron reflectivity has been used to characterize the structures and pH response of the brushes. The polymer brushes are well-described by Gaussian density profiles with an additional thin, dense layer close to the solid-liquid interface for the thicker brushes at pH 7 and 9, which produces a spike in the density profile. The spike in the distribution accounts for less than 5% of the polymer and disappears as the brushes swell at pH 3. The observed swelling behavior has been used in combination with the predictions of scaling theory and previous experimental measurements to determine the grafted density of PDMAEMA chains.

Surface-initiated atom transfer radical polymerization (SI-ATRP) has been used to grow brushes of poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) from silicon wafers using a polyelectrolytic macroinitiator on planar silicon wafers. Film thicknesses of up to 450 nm were possible within 21 h, and the effect of adding activator and deactivator species on the brush growth rate was studied. The solvation of PMPC brushes in mixed alcohol/water solvents was investigated using in situ ellipsometry. Co-nonsolvency (a re-entrant swelling transition) behavior was observed in water/ethanol binary mixtures; that is, the PMPC brushes were highly swollen in either pure ethanol or water but became deswollen at specific ethanol-rich solvent compositions. A similar effect was obtained with water/2-propanol mixtures, except that in this case pure 2-propanol was not a particularly good solvent for the PMPC chains. However, co-nonsolvency was not observed for water/methanol binary mixtures, since the brushes remained well swollen at all solvent compositions. This is consistent with prior reports of co-nonsolvency effects in both PMPC gels and linear PMPC chains. However, this is the first report of this phenomenon for PMPC brushes and one of the first examples of co-nonsolvency observed for any polymer brush system. A direct comparison of brush and gel swelling reveals an approximate power-law relationship between the equilibrium volumes of these two systems at various solvent compositions, which is interpreted by treating the brush layer as a surface-attached gel. We believe this to be the first quantitative comparison of brush and gel swelling using the same polymer under the same conditions. The kinetics of the PMPC brush response to adjustment of the alcohol/water composition is relatively fast, with the brush volume change occurring on time scales of less than 1 min as judged by in situ ellipsometry.

This paper presents measurements, using the surface force balance (SFB), of the normal and shear forces in aqueous solutions between polyelectrolyte layers grown directly on mica substrates (grafted-from). The grafting-from was via surface-initiated atom transfer radical polymerization (surface-initiated ATRP) using a positively charged methacrylate monomer. X-ray reflectometry measurements confirm the successful formation of polyelectrolyte layers by this method. Surface-inititated ATRP has the advantages that the polymer chains can be strongly grafted to the substrate, and that high grafting densities should be achievable. Measured normal forces in water showed a long-range repulsion arising from an electrical double layer that extended beyond the polyelectrolyte layers, and a stronger, shorter-range repulsion when the polyelectrolyte brushes were in contact. Swollen layer thicknesses were in the range 15-40 nm. Upon addition of approximately 10(-2)-10(-1) M sodium nitrate, screening effects reduced the electrical double layer force to an undetectable level. Shear force measurements in pure water were performed, and the measured friction may arise from polymer chains bridging between the surfaces.

The layer-by-layer (L-b-L) deposition of oppositely charged polyelectrolytic macroinitiators has been demonstrated on planar silica substrates. The build-up of the macroinitiator multilayers was monitored by ellipsometry (up to 21 layers) and dual polarization interferometry (up to 17 layers) and good agreement was found between these techniques. The increase in L-b-L thickness was approximately linear, with an average thickness of 2.3 A per layer of deposited macroinitiator. Surface-initiated ATRP of a model nonionic methacrylic monomer, 2-hydroxyethyl methacrylate (HEMA) in a 1:1 methanol/water mixture was conducted at ambient temperature. Increasing the number of macroinitiator layers led to a significant increase in PHEMA brush thickness up to 110 nm, which is attributed to the greater surface grafting density. PHEMA brush thicknesses obtained after 22 h showed a linear dependence on the number of layers of deposited macro-initiator, with all layers exhibiting near-identical growth kinetics. X-ray photoelectron spectroscopy was used to monitor L-b-L assembly and also to confirm PHEMA growth. This technique indicated the loss of small counterions from the multilayers during L-b-L deposition and confirmed an increase in the surface density of bromoester initiator groups as the number of deposited macroinitiator layers was increased. For 17 macroinitiator layers, the bromoester initiator density is estimated to be approximately 4.9 +/- 0.2 nm (-2) from the DPI data. This is comparable to that calculated for ATRP initiator monolayers obtained by either thiol or silane chemistry. Ellipsometry suggested that the macroinitiator multilayers were weakly hydrated prior to the in situ HEMA polymerization. AFM studies indicated that the PHEMA brushes had appreciable surface roughness, but this roughness became negligible compared to the brush thickness with increasing macroinitiator layers.

Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.

The temperature, salt, and pH dependencies of unfolding of four recombinant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyA1 from the hyperthermophiles Methanothermus fervidus and Pyrococcus strain GB-3a) have been determined by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). The thermal unfolding of these proteins is > 90% reversible, with concentration-dependent apparent Tm values and asymmetric unfolding transitions that are fit well by a two-state unfolding model in which a histone dimer unfolds to two random coil monomers. rHPyA1 dimers are stable in the absence of salt, whereas rHMfA, rHMfB, and rHFoB dimers unfold at 20 degrees C and pH 2 in solutions containing < 200 mM, < 400 mM, and < 1.5 M KCl, respectively. rHMfA, rHMfB, and rHFoB also experience significant cold denaturation in low salt concentrations and at low pH. The midpoint of thermal unfolding of a 1 M protein solution (T degree value) and the temperature dependency of the free energy of unfolding have been established for each histone, and both parameters correlate with the growth temperature of the originating archaeon. The changes in heat capacity upon unfolding are similar for the four histones, indicating that enhanced thermostability is not obtained by altering the curvature of the stability curve. Rather, the stability curves for the histones from the hyperthermophiles are displaced vertically to higher energies and laterally to higher Tmax values relative to the stability curve for rHFoB. The maximal free energies of unfolding for rHFoB, rHMfA, rHMfB, and rHPyA1 are 7.2, 15.5, 14.6, and 17.2 kcal/mol at 32, 35, 40, and 44 degrees C, respectively. T degree values for rHFoB, rHMfA, rHMfB, and rHPyA1 are 75, 104, 113, and 114 degrees C, respectively, at pH 5 in 0.2 M KCl. Structural features within the conserved histone fold that might confer these stability differences are discussed.

The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to double-stranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA. Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound. Reverse titration data have been fit directly to the McGhee-von Hippel model [McGhee, J., & von Hippel, P. (1974) J. Mol. Biol. 86, 469-489] using nonlinear regression. Sac7d binds noncooperatively to poly(dGdC) x poly(dGdC) with an intrinsic affinity of 6.5 x 10(6) M(-1) and a site size of 4 base pairs in 1 mM KH2PO4 and 50 mM KC1 (pH 6.8). Some binding sequence preference is noted, with the binding to poly(dIdC) x poly(dIdC) over 10-fold stronger than to poly(DAdT) x poly(dAdT). The binding is largely driven by the polyelectrolyte effect and is consistent with a release of 4.4 monovalent cations from DNA upon complex formation or the formation of 5 ion pairs at the protein-DNA interface. Extrapolation of salt back-titration data to 1 M KC1 indicates a -2.2 kcal/mol nonelectrostatic contribution to the binding free energy. A van't Hoff analysis of poly(dGdC) x poly(dGdC) binding shows that the binding enthalpy is approximately zero and the process is entropically driven. The affinity decreases slightly between pH 5.4 and 8.0. There is no significant difference between the binding parameters of recombinant Sac7d and native Sac7 proteins, indicating that methylation of the native protein has no effect on the DNA binding function. The binding of Sac7d to various DNAs leads to a significant increase in the DNA long-wavelength circular dichroism (CD) band, the intensity of which shows a sigmoidal dependence on Sac7d concentration. The sigmoidal CD binding isotherm can be quantitatively modeled by a conformational transition in the DNA that is cooperatively induced when protein monomers are bound within a given number of base pairs, ranging from zero for poly(dIdC) x poly(dIdC) to 8 or less for poly(dAdG) x poly(dCdT).

The Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius are a heterogeneous mixture of small, thermostable, nonspecific DNA-binding proteins. One of these proteins, Sac7d, has been overexpressed in Escherichia coli to provide a homogeneous preparation for structure, stability, and function studies. We present here essentially complete sequence-specific 1H NMR assignments for Sac7d, a delineation of secondary structural elements, and the high-resolution solution structure obtained from a full relaxation matrix refinement. The final structure provides an excellent fit to the NMR data with an NOE R-factor of 0.27 for backbone NOEs. The structure has a compact globular fold with 82% of the sequence involved in regular secondary structure: an antiparallel two-stranded beta-ribbon with a tight turn, followed by a short 3(10) helix, an antiparallel three-stranded beta-sheet, another short 3(10) helix, and finally four turns of alpha-helix. The amphipathic alpha-helix packs across the hydrophobic face of the three-stranded beta-sheet in an open-faced sandwich arrangement with at least one turn of the helix exposed beyond the sheet. The hydrophobic face of the beta-ribbon packs against a corner of the twisted beta-sheet. The single tryptophan responsible for the 88% fluorescence quenching upon DNA binding is exposed on the surface of the three-stranded beta-sheet. Lysines 5 and 7, whose monomethylation may be associated with enhanced thermostability, are highly solvent exposed along the inner edge of the two-stranded ribbon. The structure of Sac7d differs in many respects from that reported for the homologous native Sso7d [Baumann et al. (1994) Nature Struct. Biol. 1, 808] with a backbone RMSD greater than 3.0 A, largely due to the packing and length of the C-terminal alpha-helix which may be important in Sac7d DNA binding.

The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced. The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively. Southern blots indicate that these are the only two Sac7 protein genes in S. acidocaldarius, each present as a single copy. Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species. The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements. Potential ribosome binding sites have been identified downstream of the initiator codons. The sac7d gene has been expressed in E. coli, and various physical properties of the recombinant protein have been compared with those of native Sac7. The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds. The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly[dGdC].poly[dGdC] in 0.01 M KH2PO4 at pH 7.0. The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein. Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C.

A simple procedure is introduced for accurately defining the precision with which the Cartesian coordinates of any macromolecular structure are determined by nuclear Overhauser data. The method utilizes an ensemble of structures obtained from an array of independent simulated data sets derived from a final structure. Using the noise-free, back-calculated NOE spectrum as the "true" NOE spectrum, simulated Monte Carlo data sets are created by superimposing onto the "true" spectrum Gaussian distributed noise with a standard deviation equal to that of the residuals. Full relaxation matrix refinements of the simulated data sets provide probability distributions of the Cartesian coordinates for each atom in the model. Molecular dynamics simulations are included to estimate the effect of sparse information on the precision. The procedure is applied here to the 22-residue peptide hormone motilin, and the results are compared to those obtained using the conventional method of analyzing multiple refinements using a single distance constraint set. The average root mean square deviation for alpha-carbon atoms in the central portion (Arg12-Arg18) of the single helix of motilin was determined to be 0.72 A by the Monte Carlo method, compared to 1.3 A determined by an analysis of the 10 best DIANA structures using the same number of constraints between the same atoms. The origin of the bias of the conventional method is discussed.

A model of the structure of the 22 amino acid residue gastrointestinal peptide hormone motilin in 30% hexafluoro-2-propanol has been obtained by using distance constraints obtained from two-dimensional nuclear Overhauser enhancements. A set of initial structures have been generated by using the distance geometry program DIANA, and 10 of these structures have been refined by using restrained molecular dynamics (AMBER). The resulting structures are virtually indistinguishable in terms of constraint violations and energies and display less than 0.5-A root mean square deviations (RMSD) of the backbone atom positions from Tyr7 to Lys20. A comparison of back-calculated and experimental NOE intensities indicates that RMSD's are not the best indicators of the goodness of fit or of the precision with which the structure is defined. The structure was further refined by fitting the experimental NOE data using an iterative full relaxation matrix analysis. The mean error between the observed and calculated backbone NOE intensities for the final refined structure was 0.23 for the full length of the molecule, 0.18 for the region from Glu9 to Lys20, and 0.29 for the region from Phe1 to Gly8. R factors for the same regions were 0.27, 0.19, and 0.43, respectively. All of the NOE-determined structures consistently display an alpha-helix which extends from Glu9 to Lys20. Considerable lack of definition of structure exists at the amino and carboxyl ends of the molecule and also in the vicinity of Thr6-Tyr7-Gly8. A tendency to form a wide turn appears to exist over the sequence Pro3-Ile4-Phe5-Thr6, but the structure in this region is not well defined by the NOE data.

In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

A method is presented for determining the circular dichroism (CD) of systems whose CD spectra contain contributions from CD differential scattering. The technique is shown to detect light over 4 pi steradians, and thus, for the first time, a complete correction for scattering is possible. The method is applied to ethanol-condensed DNA and poly[d(A-C).d(G-T)]. From the results obtained, the former are proposed to have an A-type secondary structure. The condensed polynucleotide particles are shown to exhibit behavior similar to that of cholesteric liquid crystals. CD difference spectra, obtained from the scattering corrections and showing the contributions to different sections of the scattering envelope, are displayed. It is asserted that these scattering patterns contain information about the tertiary structure of the condensed DNA particles studied.

We have measured the isotropic absorption (Aiso) and linear dichroism (LD) of Escherichia coli DNA in 0.01 M Na+ (10.4 base pairs per turn of B form), 5.5 M NH4F (10.2 base pairs per turn of B form), and 80% trifluoroethanol (A form) into the vacuum UV spectral region. The reduced dichroism spectrum (LD divided by Aiso) of DNA in the A conformation differed from those of the B conformations, demonstrating that LD is a sensitive method for distinguishing DNA conformation. The reduced dichroism spectra of the B conformations were similar, indicating little change in the orientation of the bases for DNA in high salt. The wavelength dependence of the reduced dichroism indicates that the angle between the base planes and the helix axis is less than 76 degrees for all three conformations of DNA.