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Solution structure of oxidized rat microsomal cytochrome b5 in the presence of 2 M guanidinium chloride: monitoring the early steps in protein unfolding.

One- and two-dimensional proton NMR spectroscopy has been employed in order to study the denaturation effect of guanidinium chloride (GdmCl) on the oxidized state of the A-form of rat microsomal cytochrome b5 (cyt b5). The protein rapidly starts losing the heme at denaturant concentrations larger than approximately 2.0 M and a largely unfolded protein is eventually obtained. An estimate of the unfolding kinetics is obtained and, by use of a two-state model (folded left and right arrow unfolded), a value for DeltaG degrees. Below this concentration, small (</=0.15 ppm) but systematic chemical shift variations take place for the diamagnetic as well as the hyperfine-shifted signals, indicating that some structural changes occur. However, the protein core maintains its overall structure. The analysis of the two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY) maps has allowed the determination of the solution structure of the protein in the presence of 2 M GdmCl. By use of 1199 meaningful NOESY constraints (obtained from the assignment of 75% of the total protons) and 166 pseudocontact shifts, a family of 40 structures has been obtained through the program PSEUDYANA. The family was further refined through restrained energy minimization and the final root mean square deviation (RMSD) values with respect to the average structure are 0.67 +/- 0.10 A and 1.14 +/- 0.11 A for the backbone and heavy atoms, respectively. The quality of the present structure is equivalent to that of the one obtained recently for the native form [Arnesano et al. (1998) Biochemistry 37, 173-184], thus allowing a meaningful comparison between the two structures. Upon addition of 2 M GdmCl, significant local structural differences are induced to the protein backbone segments comprising residues 33-38 (helix alpha2) and 62-64 (end of helix alpha4-beginning of helix alpha5) while the overall folding scheme of the protein is still maintained. These protein regions form part of the "pocket" supporting the heme, whose plane is also rotated by approximately 10 degrees around an axis connecting the C2 and C8 carbon atoms. The initial steps of the unfolding process involve breaking of a few hydrogen bonds that stabilize local structural conformations. The hydrogen bond between Ser 64 and propionate 7, which stabilizes the heme binding to the protein frame, is broken in the presence of 2 M GdmCl. The same occurs for two hydrogen bonds between two beta-strands (beta2 and beta3), thus inducing the disruption of one of the antiparallel beta-sheets forming one side of the heme cavity. Our results are critically discussed in connection with the native-state protein local backbone mobility characteristics and point to the backbone carbons of Glu 37 and Ser 64 being the first "breaking points" of the protein frame once the global unfolding reaction is initiated at a somewhat higher concentration of denaturant.

DNA sequence-specific recognition by the Saccharomyces cerevisiae "TATA" binding protein: promoter-dependent differences in the thermodynamics and kinetics of binding.

The equilibrium binding and association kinetics of the Saccharomyces cerevisiae TATA Binding Protein (TBP) to the E4 and Major Late promoters of adenovirus (TATATATA and TATAAAAG, respectively), have been directly compared by quantitative DNase I titration and quench-flow "footprinting". The equilibrium binding of TBP to both promoters is described by the equilibrium TBP + DNA"TATA" left and right arrow TBP-DNA"TATA". The salt dependence of TBP binding to both promoters is identical within experimental error while the temperature dependence differs significantly. The observed rate of association follows simple second-order kinetics over the TBP concentration ranges investigated. The salt and temperature dependencies of the second-order association rate constants for TBP binding the two promoters reflect the dependencies determined by equilibrium binding. The TBP-E4 promoter interaction is entropically driven at low temperature and enthalpically driven at high temperature while the TBP-Major Late promoter reaction is entropically driven over virtually the entire temperature range investigated. These data suggest that the reaction mechanisms of TBP-promoter interactions are TATA sequence-specific and provide for differential regulation of promoters as a function of environmental variables.

Phylogenetic Analysis of Reptilian Hemoglobins: Trees, Rates, and Divergences

Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated alpha chains into alphaA and alphaD types, which are present in all reptiles except crocodiles, where only alphaA chains are expressed. The occurrence of the alphaD chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of beta chains (betaI and betaII), while only one type of beta chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both alpha and beta sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon + squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate alphaA and alphaD chains and crocodilian beta chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (alphaA) and 3.3 (beta) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate left and right arrow sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved approximately 3.5 (alphaA) or approximately 1.7 times (beta) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times.

Kinetics and Mechanism for Formation of Olefin Complexes in the Reaction between Palladium(II) and Maleic Acid

Complex formation between Pd(H(2)O)(4)(2+) and maleic acid (H(2)A) has been studied at 25 degrees C and 2.00 M ionic strength in acidic aqueous solution. Reaction takes place with 1:1 stoichiometry. The kinetics has been followed by use of stopped-flow spectrophotometry under pseudo-first-order conditions with maleic acid in excess. In the concentration ranges 0.01 </= [H(2)A](tot) </= 0.50 M and 0.40 </= [H(+)] </= 2.00 M, kinetic traces are biphasic. The biphasic kinetics and the dependence of reaction rate on pH and maleic acid concentration are rationalized in terms of a complex reaction mechanism of the type A right arrow over left arrow B --> C where, in addition, both steps contain contributions from parallel reactions. The amplitude of the first phase increases with increasing [H(2)A](tot) and with decreasing [H(+)]. Multiwavelength global analysis of the kinetic traces and the UV-vis spectral changes suggest that a monodentate oxygen-bonded hydrogen maleate complex, [Pd(H(2)O)(3)OOCCH=CHCOOH](+), B, with stability constant K(2) = 205 +/- 40 M(-)(1) is formed as an intermediate in this first step via two parallel reversible reactions in which Pd(H(2)O)(4)(2+) reacts with maleic acid and hydrogen maleate, respectively. In the following step, B --> C, slow intramolecular ring closure with a rate constant of 0.8 +/- 0.1 s(-)(1) at 25 degrees C gives the reaction product C, which is concluded to be a 4.5-membered olefin-carboxylato chelate complex on the basis of stoichiometry and UV-vis/NMR spectra. Parallel and irreversible attack by maleic acid and hydrogen maleate acting as olefins on the intermediate B also leads to formation of C. C is stable for at least 20 h for concentrations of </=2 mM. Global multiwavelength analysis and simulations show that accumulation of the intermediate B is between ca. 2% and 60% depending on pH and concentration of maleic acid. Neither a steady-state approximation nor a rapid preequilibrium assumption can be used for the kinetics data treatment. Exact rate expressions for the fast and slow phases have been used to derive all rate constants involved. Olefins are inefficient nucleophiles toward palladium(II), even less efficient than carboxylic acids and carboxylates.

Strategies to target kyotorphin analogues to the brain.

The design, synthesis, and pharmacological evaluation of brain-targeted chemical delivery systems (CDS) for a kyotorphin analogue (Tyr-Lys) are described. The brain-targeted compound contains the active peptide in a packaged, disguised form, flanked between the lipophilic cholesteryl ester on the C-terminus and the 1, 4-dihydrotrigonellyl redox targetor, attached to the N-terminus through strategically selected L-amino acid(s) spacer. It was found that for successful brain targeting, the epsilon-amine of Lys needs to be also converted to a lipophilic function. Through sequential enzymatic bioactivation, the Tyr-Lys dipeptide is released in a sustained manner, producing significant and prolonged analgesic activity as demonstrated by the rat tail latency test. An alternate strategy was also employed. Lys was replaced by a redox amino acid pair, Nys+ left and right arrow Nys, the nicotinamide left and right arrow 1,4-dihydronicotinamide analogues of Lys (Nys+ is 2-amino-6-(3-carbamoyl-1-pyridiniumyl)hexanoic acid). The Nys form is lipophilic and facilitates delivery in addition to the C- and N-terminal lipophilic functions. Enzymatic oxidation to Nys+ provides the lock-in, followed by removal of the lipophilic groups, releasing Tyr-Nys+ from the brain-targeted analogue (BTRA). Nys+ was shown to be an effective substitution for Arg or Lys. The activities of the CDS and BTRA, respectively, were antagonized by naloxone, supporting the designed brain-targeted processes. The most potent compound is the two-proline spacer containing CDS (CDS-PP), followed by the BTRA.

Changes in Steady-state Conformational Equilibrium Resulting from Cytoplasmic Mutations of the Na,K-ATPase α-Subunit

Mutations comprising either deletion of 32 amino acids from the NH2 terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase result in a shift in the steady-state E1 left arrow over right arrow E2 conformational equilibrium toward E1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant alpha1M32E233K double mutant, growth was markedly reduced unless the K+ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (approximately 10-fold) K+ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in Vmax and a compensatory approximately 3-fold increase in expressed alpha1M32E233K protein. In contrast to the behavior of either alpha1M32 or E233K, alpha1M32E233K also showed alterations in apparent cation affinities. K'Na was decreased approximately 2-fold and K'K was increased approximately 2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233 substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.

Gastrointestinal Monitoring Using Measurement of Intramucosal PCO2

Gastrointestinal Monitoring Using Measurement of Intramucosal PCO2

Detection of atomic deuterium in the upper atmosphere of Mars.

High-resolution spectroscopy of Mars' atmosphere with the Hubble Space Telescope revealed the deuterium Lyman alpha line at an intensity of 23 +/- 6 rayleighs. This measured intensity corresponds to HD/H2 = 1.5 +/- 0.6 x 10(-4), which is smaller by a factor of 11 than HDO/H2O. This indicates that fractionation of HD/H2 relative to that of HDO/H2O is not kinetically controlled by the rates of formation and destruction of H2 and HD but is thermodynamically controlled by the isotope exchange HD + H2O left and right arrow HDO + H2. Molecular hydrogen is strongly depleted in deuterium relative to water on Mars because of the very long lifetime of H2 (1200 years). The derived isotope fractionation corresponds to an estimate of a planetwide reservoir of water ice about 5 meters thick that is exchangeable with the atmosphere.

Investigation of CO binding and release from Mo-nitrogenase during catalytic turnover.

During enzymatic turnover in the presence of CO, Mo-nitrogenase has been shown to generate two different EPR signals termed lo-CO (PCO = 0.08 atm) and hi-CO (PCO = 0.5 atm). When the formation of hi-CO is monitored under the conditions of very low electron flux, a 2 min lag is observed prior to the initial detection of the signal followed by a near-linear rate of formation during which the S = 3/2 cofactor signal exhibits similar decay kinetics. Increasing the electron flux produces a significant increase in the rate of both the formation of hi-CO and the decay of the S = 3/2 cofactor. These results are interpreted in terms of a state of the enzyme (redox or structural) generated only during turnover which is needed to initially bind CO to the cofactor. Under high electron flux conditions, new EPR inflections are observed at g = 5.78, 5.15 and g = 1.95, 1.81 and tentatively assigned to S = 3/2 and 1/2 states of the CO-bound cofactor and 1 equiv of oxidized P cluster, respectively. Sudden removal of CO from the environment results in the slow decay (>10 min) of both the hi-CO signal and CO inhibition of acetylene reduction activity. The use of ethylene glycol to quench enzymatic activity strongly inhibits the decay of hi-CO (in the presence of CO) and the subsequent decay of lo-CO (after removal of CO) but does not prevent the reversible interconversion hi-CO left and right arrow lo-CO + CO.

Ruthenium Nitrosyl Complexes with N-Heterocyclic Ligands.

A new route was developed for preparing a series of trans nitrosyl complexes of general formula trans-[Ru(NH(3))(4)L(NO)](BF(4))(3), where L = imidazole, L-histidine, pyridine, or nicotinamide. The complexes have been characterized by elemental analysis, molar conductance measurements, UV-visible, infrared, proton nuclear magnetic, and electron paramagnetic resonance spectroscopies, and electrochemical techniques. The compounds possess relatively high nu(NO) stretching frequencies indicating that a high degree of positive charge resides on the coordinated nitrosyl group. The nitrosyl complexes react with OH(-) according to the equation trans-[Ru(NH(3))(4)L(NO)](3+) + 2OH(-) right arrow over left arrow trans-[Ru(NH(3))(4)L(NO(2))](+) + H(2)O, with a K(eq) (at 25.0 degrees C in 1.0 mol/L NaCl) of 2.2 x 10(5), 5.9 x 10(7), 9.7 x 10(10), and 4.6 x 10(13) L(2) mol(-)(2) for the py, nic, imN, and L-hist complexes, respectively. Only one redox process attributed to the reaction [Ru(II)(NH(3))(4)L(NO(+))](3+) + e(-) right arrow over left arrow trans-[Ru(II)(NH(3))(4)L(NO(0))](2+) was observed in the range -0.45 to 1.20 V for all the nitrosyl complexes. Linear correlations are observed in plots of nu(NO) versus E(1/2) and of E(1/2) versus summation operatorE(L) showing that the oxidizing strength of the coordinated NO increases with increase in L pi-acidity. The crystal structure analysis of trans-[Ru(NH(3))(4)nicNO](2)(SiF(6))(3) shows that the mean Ru-N-O angle is very close to 180 degrees (177 +/- 1 degrees ) and the mean N-O distance is 1.17 +/- 0.02 Å, thus confirming the presence of the Ru(II)-NO(+) moiety in the nitrosyl complexes studied.

Effects of forced expression of an NH2-terminal truncated beta-Catenin on mouse intestinal epithelial homeostasis.

beta-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway and as an effector of cell-cell adhesion through its association with cadherins. To explore the in vivo effects of beta-catenin on proliferation, cell fate specification, adhesion, and migration in a mammalian epithelium, a human NH2-terminal truncation mutant (DeltaN89 beta-catenin) was expressed in the 129/Sv embryonic stem cell-derived component of the small intestine of adult C57Bl/6-ROSA26 left and right arrow 129/Sv chimeric mice. DeltaN89 beta-Catenin was chosen because mutants of this type are more stable than the wild-type protein, and phenocopy activation of the Wnt/Wingless signaling pathway in Xenopus and Drosophila. DeltaN89 beta-Catenin had several effects. Cell division was stimulated fourfold in undifferentiated cells located in the proliferative compartment of the intestine (crypts of Lieberkühn). The proliferative response was not associated with any discernible changes in cell fate specification but was accompanied by a three- to fourfold increase in crypt apoptosis. There was a marked augmentation of E-cadherin at the adherens junctions and basolateral surfaces of 129/Sv (DeltaN89 beta-catenin) intestinal epithelial cells and an accompanying slowing of cellular migration along crypt-villus units. 1-2% of 129/Sv (DeltaN89 beta-catenin) villi exhibited an abnormal branched architecture. Forced expression of DeltaN89 beta-catenin expression did not perturb the level or intracellular distribution of the tumor suppressor adenomatous polyposis coli (APC). The ability of DeltaN89 beta-catenin to interact with normal cellular pools of APC and/or augmented pools of E-cadherin may have helped prevent the 129/Sv gut epithelium from undergoing neoplastic transformation during the 10-mo period that animals were studied. Together, these in vivo studies emphasize the importance of beta-catenin in regulating normal adhesive and signaling functions within this epithelium.

Phosphorylation Destabilizes the Amino-Terminal Domain of Enzyme I of the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System

Thermal stabilities of enzyme I (63 562 M(r) subunit, in the Escherichia coli phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), and a cloned amino-terminal domain of enzyme I (EIN; 28 346 Mr) were investigated by differential scanning calorimetry (DSC) and far-UV circular dichroism (CD) at pH 7.5. EIN expressed in a delta pts E. coli strain showed a single, reversible, two-state transition with Tm = 57 degrees C and an unfolding enthalpy of approximately 140 kcal/mol. In contrast, monomeric EIN expressed in a wild-type strain (pts+) had two endotherms with Tm congruent with 50 and 57 degrees C and overall delta H = 140 kcal/mol and was converted completely to the more stable form after five DSC scans from 10 to 75 degrees C (without changes in CD: approximately 58% alpha-helices). Thermal conversion to a more stable form was correlated with dephosphorylation of EIN by mass spectral analysis. Dephospho-enzyme I (monomer right arrow over left arrow dimer) exhibited endotherms for C- and N-terminal domain unfolding with Tm = 41 and 54 degrees C, respectively. Thermal unfolding of the C-terminal domain occurred over a broad temperature range ( approximately 30-50 degrees C), was scan rate- and concentration-dependent, coincident with a light scattering decrease and Trp residue exposure, and independent of phosphorylation. Reversible thermal unfolding of the nonphosphorylated N-terminal domain was more cooperative, occurring from 50 to 60 degrees C. DSC of partially phosphorylated enzyme I indicated that the amino-terminal domain was destabilized by phosphorylation (from Tm = 54 to approximately 48 degrees C). A decrease in conformational stability of the amino-terminal domain of enzyme I produced by phosphorylation of the active-site His 189 has the physiological consequence of promoting phosphotransfer to the phosphocarrier protein, HP(r).

Interactions between Fragments of Trypsinized Na,K-ATPase Detected by Thermal Inactivation of Rb+ Occlusion and Dissociation of the M5/M6 Fragment

This work provides evidence for interactions between fragments of "19-kDa membranes," a trypsinized preparation of Na,K-ATPase that retains cation occlusion and ouabain binding. Previously, we reported rapid thermal inactivation of Rb+ occlusion at 37 degreesC (Or, E., David, P., Shainskaya, A., Tal, D. M., and Karlish, S. J. D. (1993) J. Biol. Chem. 268, 16929-16937). We describe here the detailed kinetics of thermal inactivation. In the range 25-35 degreesC, a two-step model (N left and right arrow U --> I, where N is the native species, U is the reversibly unfolded intermediate, and I is the irreversibly denatured form) fits the data. Reversibility of inactivation has been observed at 25 degreesC, consistent with the model. At 37 degreesC and higher temperatures, the data can be fitted to the simple mechanism N --> I, i.e. U is not significant as an intermediate. Occluded cations (Na+, Rb+, K+, Tl+, NH4+, and Cs+) and ouabain protect strongly against thermal inactivation. Ca2+, Ba2+, and La3+ ions do not protect. Proteolysis experiments provide independent evidence that disorganization can occur in stages, first in transmembrane segments and then in extra-membrane segments of the fragments. Analysis of selective dissociation of the M5/M6 fragment at 37 degreesC (Lutsenko, S., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940), using a specific antibody, showed that inactivation of Rb+ occlusion precedes dissociation of the fragment, and only approximately 50% of the fragment is released when occlusion is fully inactivated. In the presence of Ca2+ ions, occlusion is inactivated, but the M5/M6 fragment is not released. The experiments demonstrate that occlusion is inactivated by disruption of interactions between fragments of 19-kDa membranes, and only then does the M5/M6 fragment dissociate. Interactions between the M5/M6 and M7/M10 fragments seem to be essential for maintenance of Rb+ occlusion.

Verapamil block of the delayed rectifier K current in chick embryo dorsal root ganglion neurons.

We have used the patch-clamp method in the whole-cell configuration to investigate the mechanism of block of the delayed rectifier K current (IDRK) by verapamil in embryonic chick dorsal root ganglion (DRG) neurons. Verapamil induced a dose-dependent decay of the current, without altering its activation kinetics. This observation, together with the good description of IDRK time course at various blocker concentrations with the computer simulation of a three-state chain model (closed left and right arrow open left and right arrow open-blocked), indicates that verapamil acts as a state-dependent, open-channel blocker. To account for the double-exponential time course of recovery from block, this minimal kinetics scheme was expanded to include a closed-blocked state resulting from channel closure (at hyperpolarized voltages) with verapamil still bound to it. The apparent block and unblock rate constants assessed from verapamil-induced current decay in the presence of external Na were 0.95 +/- 0.05 ms-1mM-1 and 0.0037 +/- 0.0016 ms-1, respectively. When external Na was replaced by K, only the unblock rate constant changed, to 0.02 +/- 0.009 ms-1. Under these ionic conditions it was also observed that the recovery from block was modified from the double-exponential time course in the presence of external Na (tau1 = 160 ms; tau2 = 1600 ms), to a faster single-exponential recovery (tau = 100 ms). We tested the voltage dependence of block by applying stimulation protocols aimed at eliminating bias easily introduced by the shift of the gating equilibrium and by the coupling of channel activation and block. Under these experimental conditions the resulting block rate constant was not measurably voltage dependent.

Carbon Nanotube Tips: High-Resolution Probes for Imaging Biological Systems

Herein, we report the first atomic force microscopy (AFM) imaging studies with carbon nanotube tips that address their potential to improve lateral resolution and to probe biological systems. Multiwall carbon nanotubes (MWNTs) and single-wall carbon nanotubes (SWNTs) have been attached to the ends of single-crystal silicon cantilever tip assemblies and used to image amyloid â(1-40) derived protofibrils and fibrils by tapping mode AFM. Image analysis shows that the average resolution obtained with the nanotube tips is significantly better than that obtained with our best silicon tips and provides new insight into the structure and assembly mechanism of amyloid fibrils. The potential for imaging with molecular resolution using these tips is discussed. The feature resolution obtained by AFM is determined in large part by the size and shape of the probe tip used for imaging.1,2 Commercially available probes consist of microfabricated pyramids of Si or Si3N4 that have end radii of curvature as small as 10 nm but are often much larger.2,3 These tips place significant constraints on potential lateral resolution, and furthermore, the pyramidal shape restricts the ability of these tips to access narrow and deep features. Small cone angle carbon protrusions with radii of 10-30 nm deposited on the ends of conventional tips significantly improve depth but not lateral resolution.4 Recently, a potential breakthrough in probe technology was achieved by attaching MWNTs to the ends of Si tips.5 In this work, the cylindrical geometry of the MWNT tips was exploited to image a deep grating with excellent fidelity, although potential improvements in lateral resolution were not investigated. A typical scanning electron microscopy (SEM) image of a MWNT tip attached to a conventional single-crystal silicon cantilever tip assembly is shown in Figure 1.5,6 This MWNT tip extends ca. 1.8 μm from the end of the pyramidal Si tip to which it is attached. The diameter of the major portion of the nanotube tip, 100 nm, is much larger than a single MWNT and corresponds to a bundle of tightly packed tubes.7 The MWNT bundles attached to the Si probes have diameters of 75 ( 27 nm; attached SWNT bundles8 typically are smaller with diameters of 45 ( 8 nm. The high aspect ratio nanotube tips have obvious advantages for probing deep crevices and steep features.5 Additionally, it is known that nanotubes elastically buckle above a critical force.5,9 Buckling limits the maximum force applied to a sample,10 which can prevent damage to delicate organic and biological samples, and at the same time makes the tips very robust.9 To explore the resolution of these tips and their applicability to high-resolution imaging of biological samples, we have investigated amyloid-â 1-40 (Aâ40) fibrils produced in vitro. Aâ fibrils are the primary constituent of the insoluble core of amyloid plaques that are characteristic of Alzheimer’s disease.11 Previous AFM studies have shown that Aâ initially forms protofibril structures12,13 and subsequently larger fibrils;12-14 branching of fibrils has also been observed.13,14 A typical image15 of an Aâ fibril obtained with a MWNT tip (Figure 2) shows higher resolution than observed in previous AFM studies of the fibrils. Key features include (1) a fibril Y-branch (left arrow) with the

Solvent composition and viscosity effects on the kinetics of CO binding to horse myoglobin.

Ligand binding to myoglobin in aqueous solution involves two kinetic components, one extramolecular and one intramolecular, which have been interpreted in terms of two sequential kinetic barriers. In mixed solvents and sub-zero temperatures, the outer barrier increases and the inner barrier splits into several components, giving rise to fast intramolecular recombination. The nature of these barriers and their relation to structural relaxation are examined using the effect of solvent composition and viscosity on the kinetics of CO binding to horse myoglobin in 60% ethylene glycol/water, 75% and 90% glycerol/water, 80% and 92% sucrose/water solutions. Measurements of the corresponding solvent structural relaxation rates by frequency resolved calorimetry allow us to discriminate between solvent composition and viscosity-related effects. The outer kinetic barrier controlling ligand entry and release depends on the viscosity consistent with Kramers-Stokes law of activated escape in the presence of friction. At high cosolvent concentration, we observe deviations from Stokes law, implying a smaller microviscosity at the protein-solvent interface as compared to the bulk. The inner barrier and its coupling to structural relaxation appears to be independent of viscosity but changes with solvent composition. As a possible explanation, we discuss the role of distal water molecules in the formation of the effective inner barrier. At low temperatures, this barrier has a distributed height, depending only slightly on the nature of the cosolvent and temperature at low cosolvent concentrations. In contrast, myoglobin embedded in a sucrose glass (92% sucrose/water) exhibits a temperature-dependent and bimodal enthalpy distribution. This result demonstrates that the exchange between protonation states of His64, A0 left and right arrow A1, can take place in the glass and at temperatures as low as 80 K.

The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods.

The enzyme superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide radical, is present in the cytosol and mitochondria of all oxygen-respiring eukaryotes. The cytosolic form contains copper and zinc (CuZnSOD), whereas the mitochondrial form contains manganese (MnSOD). The latter protein is synthesized in the cytosol as a MnSOD precursor, containing an N-terminal mitochondrial-targeting sequence. CuZnSOD is sensitive toward cyanide (CN) and hydrogen peroxide (H2O2), but MnSOD is not. Assays for SOD activity in cytosol from the hepatopancreas of the blue crab, Callinectes sapidus, showed the presence of a CN/H2O2-insensitive form of SOD. No CN/H2O2-sensitive CuZnSOD was found. This unexpected phenomenon was shown to occur in all decapod crustacea (crabs, lobsters, shrimp) examined. The cytosolic and mitochondrial SODs of C. sapidus were purified by means of ion-exchange, size-exclusion, and reverse-phase HPLC. The cytosolic SOD is a homodimeric protein, which exists in a monomer-dimer equilibrium (24 kDa left and right arrow 48 kDa). The protein contains approximately 1 Mn per subunit. No copper or zinc is present. Amino acid sequence analysis identified the novel cytosolic SOD as a MnSOD precursor with an abnormal mitochondrial-targeting sequence. The mitochondrial SOD of C. sapidus is similar to the MnSOD found in other eukaryotes. N-Terminal amino sequences of mitochondrial and cytosolic blue crab MnSOD differ in several positions. The MnSODs are thus encoded for by two different genes. The paradigm that all eukaryotes contain intracellular CuZnSOD and that MnSOD occurs exclusively in the mitochondria appears not to apply to a large group of marine arthropods.

Phosphoryl Group Exchange between ATP and ADP Catalyzed by H+-ATPase from Oat Roots.

ATP-ADP exchange was estimated in the presence of plasma membrane H+-ATPase of oat (Avena sativa) roots partially purified with Triton X-100 by measuring [14C]ATP formation from [14C]ADP. Most studies were done at 0[deg]C. At pH 6.0 the exchange showed: (a) Mg2+ requirement with a biphasic response giving maximal activity at 152 [mu]M and (b) insensitivity to ionic strength, [Na+], and [K+]. ATP and ADP dependence were analyzed with a model in which nucleotide-enzyme interactions are at rapid-random equilibrium, whereas E1ATP [left right arrow] E1P-ADP transitions occur in steady state. The results indicated competition between ADP and ATP for the catalytic site, whereas ATP interaction with the ADP site was extremely weak. At 0[deg]C the exchange showed a 3-fold pH increase, from pH 5.5 to 9.0. At an alkaline pH the reaction was not affected by sodium azide and carbonyl cyanide p-trifluometoxyphenyl-hydrazone, had a biphasic response to Mg2+ (maximal at 513 [mu]m), and was insensitive to ionic strength. At 20[deg]C ATP-ADP exchange was pH insensitive. At both temperatures ATP hydrolysis displayed a bell-shaped response, with a maximum around pH 6.0 to 6.5. Because no adenylate kinase activity was detected under any condition, these results demonstrate the existence of an ATP-ADP exchange reaction catalyzed by the plant H+-ATPase.

Chemical Bond Analysis of Nonlinearity of Urea Crystal

A novel and quantitative study on structure-property relationships has been carried out in urea crystal, based on the dielectric theory of complex crystals and the modified Levine bond charge model, mainly from the chemical bond viewpoint. For the first time, it was treated like this, and the bond parameters and linear and nonlinear characteristics of constituent chemical bonds were presented quantitatively. The theoretical result agrees satisfactorily with the experimental datum and can reasonably explain the nonlinear origin of urea crystal, that is, the C-N bond in the conjugated system of bonds O double left arrow C<--N-H. At the same time, the novel method should be a useful tool toward the future development of the search for new nonlinear optical (NLO) materials in the organic crystal field.

The ATP-activated Hexameric Helicase of Bacteriophage T4 (gp41) Forms a Stable Primosome with a Single Subunit of T4-coded Primase (gp61)

We have examined the formation of the primosome subassembly of the bacteriophage T4-coded DNA replication (elongation) complex from its helicase, primase, and DNA components. Previously, we had shown that the T4 helicase (gp41) exists in solution in a stable monomer left and right arrow dimer equilibrium at physiological protein (and salt) concentrations and forms a hexamer upon activation by ATP (or GTP) binding (Dong, F., Gogol, E. P., and von Hippel, P. H.(1995) J. Biol. Chem. 270, 7462-7473). Here we report that the T4 primase (gp61) is a monomer in solution under the same conditions, and that the ATP-activated helicase binds to a single gp61 primase molecule on appropriate DNA templates to reconstitute a stable primosome. We show that: (i) the gp41 helicase alone does not form a stable complex with DNA templates, although this helicase by itself can carry out moderately processive ATP-driven translocation along single-stranded DNA (Young, M. C., Schultz, D. E., Ring, D., and von Hippel, P. H.(1994) J. Mol. Biol. 235, 1447-1458); (ii) the primase alone does form a stable complex with DNA; (iii) the helicase can bind to the primase-DNA complex in the presence of ATP or GTP to form a stable ternary complex; (iv) this complex consists of six helicase subunits and one primase subunit; and (v) the reconstituted primosome is stable for at least 10 to 20 min after NTP cleavage and dissociation of the hydrolysis products. These results strongly suggest that the functional T4 DNA replication primosome consists of an integrated 6:1 helicase-primase complex bound to DNA, and that the ATP-activated helicase hexamer remains intact throughout the processive DNA replication process.