bluuebunny/crossref_metadata_2025 huggingface dataset:
Generalized Locking Profile for Angular Particles ‐ Locked Particle Theory
Authors: Meloy Thomas P. , Clark Nigel N.
Published: May 2005

Locked particles are defined as those comminuted or particles which contain both gangue and target material. By considering the shape of the comminuted particles and assuming their random distribution in the ore body prior to comminution, it is possible to derive locking profiles which give the frequency of occurrence of particles which have a given fraction of their volume occupied by target material. For most particle shapes (spheres, angular particles, angular plates and rods) the locking profile is a deep U‐shaped curve. This suggests that few particles by number contain about 50% target material while a far greater number contain either very little or very much target material. Such a conclusion is important in planning separation processes for locked particles in the mineral industry.
Fractional Penetrations for Electrostatically Charged Fibrous Filters in the Submicron Particle Size Range
Authors: Lathrache Reda , Fissan Heinz
Published: May 2005

Particle penetrations through commercially available, electrically charged fibrous filters have been measured. The particles were monodisperse and in charge equilibrium. Tests were conducted for several particle sizes in the range 0.02 μm ≤ x ≤ 1 μm. The face velocity was varied in the range of 2 cm/s to 30 cm/s. The penetration has a bimodal dependence on particle size. This behaviour is not found for uncharged filters. The reduction in penetration and bimodal dependence is attributed to electrical collection effects. As the face velocity is increased, the electrostatic collection effects are significantly reduced. The results agree quantitatively with a model for particle penetration through charged fibrous filters recently presented in the literature by the authors.
Real‐Time Ultrasonic Process Tomography of Flowing Mixtures
Authors: Hoyle Brian S.
Published: May 2005

This paper presents a generic application study of the simple but fundamental limitations in the industrial exploitation of ultrasonic process tomography to dynamic flowing mixture processes. The background to two‐dimensional ultrasonic process tomography is reviewed and the key implementation aspects which uniquely limit its performance are explored. Simple descriptors are presented, which allow the assessment of the applicability of ultrasound process tomography to particular applications.
Recent Advances of Diffraction Tomography in Geophysics, Ultrasonics, and Optics
Authors: Stamnes Jakob J. , Wedberg Torolf C.
Published: May 2005

A tutorial review of diffraction tomography is given along with an overview of recent advances of this technique in borehole geophysics, ultrasonic imaging, and optical microscopy. First, the basic principles of diffraction tomography are presented. Then, we discuss a generalized inversion algorithm, valid for irregularly spaced data and a non‐uniform background, and present reconstructions based on ultrasonic water tank data and underground georadar data. Next, we discuss a hybrid filtered backpropagation algorithm for ultrasonic and optical imaging. Quantitative images, based on experimental data, are presented of objects embedded in water or in biological tissue and probed by ultrasound, and of fibers embedded in an index matching liquid and probed by laser light.
Applications for Tomographic Technology in Mining, Minerals and Food Engineering
Authors: Williams Richard A. , Atkinson Keith , Luke Stephen P. , Keith Barlow R. , Dyer Ben C. , Smith John , Manning Michael
Published: May 2005

Existing and new applications for tomographic technology using optical, electrical radar and seismic sensing principles are described. It is demonstrated that important similarities link process instrumentation needs in the mineral benefication and food manufacturing sectors. The minerals industries provide a variety of challenging applications for tomography in the development and validation of models associated with acid mine drainage, the stability of mine workings, mineral exploration and other environmental needs such as monitoring landfill sites and groundwater plume dispersion.
Extinction Efficiency of Gradient‐index Microspheres
Authors: Kai Li , D'Alessio Antonio
Published: May 2005

The extinction efficiency of transparent spheres with a radial gradient of refractive index irradiated by electromagnetic plane waves was obtained by using the finely stratified sphere model [L. Kai, P. Massoli, Appl. Opt. 33 (1994) 501–511]. The extinction efficiency of gradient‐index microspheres and location changes of morphology‐dependent resonances on the Mie extinction efficiency curves are discussed.
The Fragmentation Fractal – a useful descriptor of broken rock
Authors: Brown Gareth J. , Miles Nick J.
Published: May 2005

The fragmentation fractal has been used to accurately describe the distribution of rock progeny from single particle breakage events. This paper illustrates the underlying physical significance of the fragmentation fractal and how it may be used as a fundamental tool in understanding rock pulverisation. The experimental determination of the fragmentation fractal is based on the measurement of particle size by project area as given by image analysis. In this format the characteristic length is defined as the square root of projected area. This approach to rock particle size analysis can have advantages over other techniques especially when the mass or number of fragments is small. A brief comparison has been made with size by projected area and the corresponding sieve size of artificial objects of different shape, but equivalent characteristic length. It is suggested that projected area measurements are less sensitive to the influence of particle shape and that this may even aid the comparison of distributions of fragments of significantly different morphologies.
Arrays of Uniform Latex Spheres as Test Objects in Optical Microscopy
Authors: Davidson John A.
Published: May 2005

It is the purpose of this communication to illustrate the utility of regular arrays of uniform high polymer latex spheres (2‐D crystals) as resolution test objects for the optical microscope. Such test specimen slides are easily prepared, inexpensive, and can be quite accurately characterized as to size of the spheres which make up the arrays.

Through their use it was possible to experimentally verify the validity of the generalized form of Abbe' s rule (see Eq. (2)) for bright field microscopy.

Similarly, it was possible to demonstrate that resolution in dark field microscopy is equal to and can sometimes surpass performance in bright field.

Array test objects also hold great promise in the teaching of microscopy. The importance of proper micros topic technique and the difference between resolution and non‐resolution, resolution and detection, and the effect of the wavelength of light on resolution are all easily demonstrated through the use of latex arrays.
The Effect of Flowagents on the Rheology of a Plastic Powder
Authors: Kaye Brian H. , Gratton‐Liimatainen Julie , Lloyd John
Published: May 2005

Flow agents are widely used in industry, such as the plastics and pharmaceutical industry, to promote the flow of cohesive powders. Few studies of the quantitative effects of the flow agents on the flow of the powder have been reported. In this communication the effect of silica flow agents on the flow of a plastic powder is studied by means of avalanching behaviour. The avalanching behaviour of the powder as modified by the silica flow agents is summarized using descriptive parameters based on the calculation of fractal dimensions in data space and phase‐space strange attractors. The structure of the strange attractors are shown to be statistically self‐similar and‐effective descriptors of the change of rheology caused by the presence of the flow agents.
Streaming Current Measuring for Determining the Zeta Potential of Granular Particles
Authors: Rodier Elizabeth , Dodds John
Published: May 2005

A simple technique is presented for determining the zeta potential of large particles ( > 20 μm) by streaming current measurements. The method has the advantage of not requiring correction for surface conductivity. The theory involves a description of pore space by either Darcy's law or the Kozeny‐Carman equation. The simplifications introduced by assimilating the pore space to a bundle of capillary tubes are discussed. Results are presented for the variation of zeta potential with pH and ionic strength for a sieve cut of spherical glass beads and two different samples of irregular particles of silicon carbide.
The Use of an Antistatic Agent to Improve Powder Flowability
Authors: Orband Jan L. R. , Geldart Derek
Published: May 2005

Small quantities of Larostat 519, an anti‐static agent, were added to four samples of a commercial organic powder which was giving flowability problems believed to be caused by electrostatics. The effects on standardised bulk densities, on the Hausner Ratio, and on the behaviour of the samples when exposed to various controlled relative humidities in an experimental rotating inclined pan, were significant, and are reported and interpreted.
Hydrodynamic Focusing of Particle Trajectories in light‐scattering counters and phase‐doppler analysers
Authors: Ovod Vladimir I.
Published: May 2005

To reduce errors when measuring sizes of particles in light‐scattering counters and phase‐Doppler analysers, the hydrodynamic focusing of particle trajectories in relation to the centre of the measurement zone is used. This focusing is effected in a special running‐type chamber. Equations describing the mechanical energy of liquid flows running through a running‐type chamber have been compiled. By solving a system of such equations, relatively simple equations for engineering calculations of the coefficient of hydrodynamic compression of particle trajectories in relation to the centre of the measurement zone have been obtained. Results of experimental research are given and the application of the proposed technique for designing improved running‐type chambers with one and two “sheaths” is demonstrated.
Studying the Avalanching Behaviour of a Powder in a Rotating Disc
Authors: Kaye Brian H. , Gratton‐Liimatainen Julie , Faddis Neil
Published: May 2005

The use of a rotating disc to study the avalanching behaviour of a powder is discussed. It is shown that the strange attractor plotted in discreet time maps summarizes useful information on the rheological behaviour of powders and powder mixtures. In particular it is shown that the avalanching behaviour is related to the particle size distribution of the powder and that one can study the changes in rheological behaviour as another powder is mixed with it. The strange attractor patterns generated are dependent upon the environmental conditions under which the experiments are carried out. For this reason the measurements are referred to as an assessment of the holistic powder rheology. The potential use of the disc to study the holistic rheology of powder systems is outlined.
Symmetrical and Asymmetrical Flow Field‐Flow Fractionation for Particle Size Determination
Authors: Schauer Thadeus
Published: May 2005

Measurements on paint components have been conducted with symmetrical flow field‐flow fractionation (sym flow FFF) and asymmetrical flow FFF (asym flow FFF). Both methods are based on the same principle of separation, but they differ in the construction of the fractionation channel and the configuration of the channel flow and crossflow. On the basis of the good selectivity and resolution of these techniques, it was possible to determine the particle size of each individual constituent of a three‐component mixture of a paint binder, pigment and filler, characterized by relatively broad and overlapping distributions, and to follow the changes on mixing them. In the case of sym flow FFF, it was possible to use thinner fractionation channels, with no system peaks and good selectivity in both the normal and steric‐hyperlayer mode. The main features of asym flow FFF are the simpler construction of the fractionation channel and the possibility of focusing the sample in the channel better and of following visually the separation. This variant should be preferably used in the normal mode of operation.
Investigations on the Behaviour of an Aerodynamic Particle Sizer and its applicability to calibrate an optical particle counter
Authors: Heidenreich Steffen , Büttner Helmut , Ebert Fritz
Published: May 2005

The calibration of an optical particle counter (OPC) by means of the Aerodynamic Particle Sizer APS (TSI, Model 3310) was investigated. The pulse‐height distribution and the aerodynamic size distribution were measured by parallel use of an OPC and the APS. A calibration curve was obtained by comparison of the two different cumulative distribution curves. First calibration results are presented for spherical particles (water droplets and glycerine droplets): A comparison of these results with Mie calculations and aerodynamic calibrations by means of sampling cyclones shows good agreement. Furthermore, measurements were carried out with non‐spherical particles. Quartz dust was used for these measurements. In order to calibrate the OPC by means of the APS, the behaviour of both devices was investigated.
Detection of Deposited Particles from the Backside of a Glass Plate
Authors: Semleit Dirk , Fissan Heinz , Yu Guangjian
Published: May 2005

Microcontamination of product surfaces by deposited particles is an important problem in clean technologies. A most sensitive product to contamination by particles is a wafer during chip production. Therefore, methods for monitoring particle deposition on wafer surfaces have been developed in the last decade. A wafer with an unstructured and reflecting surface is inserted into the process equipment. After some time, depending on the process, this wafer is removed from the process equipment and is analysed with respect to the number of deposited particles using a wafer scanner. However, in situ particle detection in a process chamber is not possible with this technique. This would be possible if, instead of a monitor wafer, a transparent glass plate is mounted, e.g. in the housing of the process equipment. Then the illuminating and scattered light detection equipment can be mounted outside the process equipment. Since both the illuminating laser beam and the scattered light have to be transmitted through the glass plate, losses will occur, which will reduce the lower limit of detection with respect to particle size. In this article we estimate the detection possibilities theoretically and experimentally.

A simple model based on Mie and vector scattering theory has been developed to describe the light‐scattering behavior of a single spherical particle on a glass plate with random surface irregularities. The scattered light of individual particles of four particle sizes (1.03, 1.6, 2.92 and 4.23 μm) on the same glass surface and from the uncontaminated area of the glass plate was measured for unpolarized and normally incident light. The values of the scattered light from this model were compared with the experimental results. The comparison shows a reasonable agreement of the angular distribution of the scattered light. The developed model is used to predict the lower limit of detection for particles on a transparent surface. The theoretical estimations show that it should be possible to detect particles of a diameter down to 0.2 μm with the described measurement technique.
Lorenz‐Mie Theory for Spheres Immersed in an absorbing host medium
Authors: Quinten Michael , Rostalski Jörn
Published: May 2005

Light scattering by particles is often used to determine velocities or concentrations of particles in gaseous or liquid streams. Within the Lorenz‐Mie theory, light scattering is well understood both for a single compact spherical particle and a single multilayered particle in a non‐absorbing surrounding medium. However, in some cases of practical importance the Lorenz‐Mie theory in its present form may fail to describe the scattering because the host medium is absorbing (e.g. water droplets in oil). In this case, a new treatment of the scattering theory is required. In previous work, solutions were obtained in the far‐field of the scattering sphere. In this paper, a rigorous solution is derived from the calculation of the total absorption rate of the particle in the host medium, which is valid for all distances from the surface of the encapsulated particle. It is shown that it is necessary to consider finite sizes R of the integrating sphere when dealing with absorbing host media. Cross‐sections are defined which are characteristic quantities not only for the particle, depending on the size of a conceptual sphere around the scatterer and the imaginary part of the refractive index of the host medium. The results obtained are discussed for the case of non‐absorbing host media and in the far‐field approximation. Some numerical examples are given which are also related to experimental results.
Optical Cross‐Sections of Soot Agglomerates: The model of the virtual refractive index and first‐order multiple scattering
Authors: Dittmann Rolf
Published: May 2005

A new technique for the calculation of the optical properties of agglomerated spherical primary particles is presented. By means of the so‐called virtual refractive index, the multiple scattering within the agglomerate is decribed by a modified Mie solution, thus avoiding restrictions with respect to the primary particle size. An additional benefit of the method is the small numerical expense required, permitting practical use for complex agglomerate structures such as real combustion aerosols. Utilizing this method, attention is focused on the calculation of the extinction cross‐section of real combustion aerosols in order to discuss the results of multiple‐wavelength extinction measurements within combustion systems. It is found that approximately the primary particle diameter is determined. Furthermore, the error that occurs can be avoided by means of the method presented without a detailed knowledge of the agglomerate size. Thus, the present work eliminates some current uncertainties inherent in optical combustion aerosol characterization.
Influence of Refractive Index Gradients within droplets on rainbow position and implications for rainbow refractometry
Authors: Anders Klaus , Roth Norbert , Frohn Arnold
Published: May 2005

Rainbow refractometry allows the determination of the refractive indices of droplets if a homogeneous refractive index can be assumed throughout the whole droplet. The influence of gradients on the angular position of the rainbow was examined for refractive index distributions with constant gradients and for distributions as they occur during cooling, heating and burning of droplets. Geometrical optics were used to determine rainbow positions. For a given value of refractive index at the surface, the rainbow position is in many cases a measure for the gradient itself. It was found that information obtained for constant gradients can often be used in cases with more complex distributions, which are found, for instance, for droplets undergoing heat‐transfer processes. Significant errors may be obtained only for a very short initial phase of the process. Experimental results obtained with monosized droplet streams, which confirm theoretical predictions of the influence of gradients on the rainbow position, are presented.
Light Reflection and Transmission by layers with oriented spheroidal particles
Authors: Berdnik Vladimir V. , Loiko Valery A.
Published: May 2005

On the basis of the numerical solution of the radiative transfer equation (RTE), the influence of non‐sphericity of oriented spheroidal particles on radiation intensity formation in a layer with a multiple scattering regime is considered. For solving the RTE with characteristics depending on the propagation direction of the radiation beam, an algorithm is developed based on the method of layer doubling. Calculations are presented of angle correlations for intensities of radiation in a layer with Fresnel reflection on the boundaries, and reflection and transmission coefficients.
Monodisperse Sprays for Various Purposes — Their production and characteristics
Authors: Brenn Günter , Durst Franz , Tropea Cameron
Published: May 2005

The production of liquid sprays represents a key technology for a wide range of industrial proceses. Most applications currently use pressure or air‐assisted atomization, resulting in the production of polydisperese sprays. Recent advances in experimental and numerical techniques for investigating liquid spraying processes, however, have enabled a closer examination of parameter optimization, leading to the conclusion that in may cases, a much narrower size distribution, or even a monodisperse spray, may exhibit many advantages. Currently monodisperse droplet generators, or drop‐on‐demand generators, do not meet this challenge of producing monodisperse sprays, primarily owing to the very low volume flow rate of liquid which is atomized. In the present work, a monodisperse sprya generator is introduced, which overcomes this difficulty for many applications.
Some Recent Developments in Twin‐Fluid Atomization
Authors: Lefebvre Arthur H.
Published: May 2005

The results of recent researches on twin‐fluid atomization are briefly reviewed, with particular emphasis on the effects of air and liquid properties on mean drop size. It is stressed that no single equation for the mean drop sizes produced in twin‐fluid atomization can satisfactorily correlate all the experimental data from any given atomizer. This is because for all types of atomizers the mechanism of liquid breakup changes from one basic mode to another with changes in atomizer operating conditions and/or liquid properties. These different modes of atomization exhibit different dependencies on atomizer dimensions and various physical properties such as liquid viscosity and air density. Equations for estimating the mean drop sizes produced in the various modes of twin‐fluid atomization are presented and discussed.
Effects of Monomer Size Distribution on the fractal dimensionality of diffusion‐limited aggregates
Authors: Chou Charles C.‐K. , Lee Chung‐Te
Published: May 2005

Computer simulations of diffusion‐limited aggregation (DLA) for monomers to investigate the effects of size and of lognormal distribution on the fractal dimensionality of the aggregates were conducted on a two‐dimensional lattice. The results show the DLA clusters posses multifractal characteristics. For clusters consisting of monodisperse monomers, the bifurcation point on the graph of the pair correlation function (PCF) for each cluster is located right at the monomers size under investigation The textural dimension (Df1) has a stable value of about 1.65, whereas the structural dimension (Df2) decreased with increase in monomer size. For the cases with monomers in log‐normal distributions, the textural dimension is around 1.67; however, the structural dimension decreases with increasing polydispersity of monomer size.
Characterising Semi‐Cohesive Powders using angle of repose
Authors: Wouters Ilse M. F. , Geldart Derek
Published: May 2005

When properly measured and interpreted angle of repose (AOR) can be a useful method of characterising a powder. Unfortunately, a multitude of definitions and measuring equipment is used. A review of the inherent problems is presented in the first part of this paper.

The second part of the paper describes the improvements made to a measuring device developed originally for measuring the angle of repose of soda ash, so that a wider range of powders can be tested. In the improved design, the tendency of cohesive and semi‐cohesive powders to block the device's funnel has been eliminated.

A total of 73 powders consisting of four different materials, and covering Groups A, A/C and C of Geldart's classification, have been tested using the new equipment. The results show that AOR increases systematically with decrease in mean particle size. Combination of AOR measured in the new device with aerated bulk density provides a quick, sensitive and effective method for characterising a wide range of powders.
Particle shape characterization using morphological descriptors
Authors: Pons Marie‐Noëlle , Vivier Hervé , Dodds John
Published: May 2005

Two descriptors based on mathematical morphology are described which permit discrimination between convex and concave polygonal shapes which often characterize crystalline particles. Their computation is based on the comparison between the silhouette of the particle and a reference shape. Three reference shapes are discussed: the convex hull, the circle circumscribed to the particle and the equivalent circle.
A fourier optics method for the simulation of measurement‐volume‐effect by the slit constraint
Authors: Qiu Hui‐He , Hsu Chin‐Tsau
Published: May 2005

A new method is introduced to analyze the accuracy of phase‐Doppler anemometry (PDA) for sizing large particles in two‐phase flows. The method is based on Fourier optics theory (FOT) and geometrical optics theory (GOT) to calculate the intensity ratio of refractive and reflective light scattered by a sphere under a slit constraint. To examine the accuracy in calculating the light intensity, the results from the GOT method were compared with those of the direct simulation based on the generalized Lorenz‐Mie theory (GLMT). This newly developed FOT/GOT method also predicts the results of phase jump due to the slit constraint. The performance of dual‐mode phase‐Doppler anemometry can also be simulated by this method.
Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons
Authors: Roder Heinrich , Wüthrich Kurt
Published: May 2005

A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin (J. Mol. Biol. 135: 199–215, 1979), which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1H NMR to provide site‐resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence‐specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the β‐sheet and the C‐terminal α‐helix of this protein, apparent refolding rates in the range from 15s−1 to 60 s−1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes.
Focusing of electric fields in the active site of Cu‐Zn superoxide dismutase: Effects of ionic strength and amino‐acid modification
Authors: Klapper Isaac , Hagstrom Ray , Fine Richard , Sharp Kim , Honig Barry
Published: May 2005

In this paper we report the implementation of a finite‐difference algorithm which solves the linearized Poisson‐Boltzmann equation for molecules of arbitrary shape and charge distribution and which includes the screening effects of electrolytes. The microcoding of the algorithm on an ST‐100 array processor allows us to obtain electrostatic potential maps in and around a protein, including the effects of ionic strength, in about 30 minutes. We have applied the algorithm to a dimer of the protein Cu‐Zn superoxide dismutase (SOD) and compared our results to those obtained from uniform dielectric models based on coulombic potentials. We find that both the shape of the protein‐solvent boundary and the ionic strength of the solvent have a profound effect on the potentials in the solvent. For the case of SOD, the cluster of positive charge at the bottom of the active site channel produces a strongly enhanced positive potential due to the focusing of field lines in the channel—a result that cannot be obtained with any uniform dielectric model. The remainder of the protein is surrounded by a weak negative potential. The electrostatic potential of the enzyme seems designed to provide a large cross‐sectional area for productive collisions. Based on the ionic strength dependence of the size of the positive potential region emanating from the active site and the repulsive negative potential barrier surrounding the protein, we are able to suggest an explanation for the ionic strength dependence of the activity of the native and chemically modified forms of the enzyme.
PseqIP: A nonredundant and exhaustive protein sequence data bank generated from 4 major existing collections
Authors: Claverie Jean Michel , Bricault Laurence
Published: May 2005

Four major protein sequence data collections (NBRF‐PIR, PSD‐Kyoto, PGtrans, and NEWAT) have been merged into a single nonredundant data bank called PseqIP. The data bank entries were automatically matched by a heuristic computer program relying on the fast computation of the number of tetrapeptides shared by two sequences. PseqIP 1.0 includes 6,068 different protein sequences for a total of 1,357,067 residues, representing most of the available sequence information to date. During the course of this work, we found about 600 occurrences course of a protein sequence recorded with a one‐amino‐acid variation in at least two different data banks. A flat file (ASCII computer‐readable format) version of PseqIP 1.0, well‐suited for exhaustive homology searches and statistical sequence analysis, is available from our laboratory.
A domain of the klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity
Authors: Freemont P. S. , Ollis D. L. , Steitz T. A. , Joyce C. M.
Published: May 2005

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3′–5′ exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3′‐5′ exonuclease active site. The larger C‐terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C‐terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3′‐5′ exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.
Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation
Authors: Shortle David , Meeker Alan K.
Published: May 2005

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m–the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration—these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild‐type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild‐type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain‐chain or chain‐solvent interactions in the denatured state have been modified—interactions which appear to involve hydrophobic groups.
Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its γ subunit and transducin
Authors: Wensel Theodore G. , Stryer Lubert
Published: May 2005

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (Tα‐GTP) is a key step invisual excitation. The finding that trypsin activates PDE (αβγ) by degrading its γ subunit and the reversal of this activation by γ led to the proposal that Tα‐GTP activates PDE by relieving an inhibitory constraint imposed by γ (Hurley and Stryer: J. Biol. Chem. 257:11094–11099, 1982). We report here studies showing that the addition of γ subunit also reverses the activation of PDE by Tα‐GTP‐γS. A procedure for preparing γ in high yield (50–80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitorya activity resides in the γ subunit. Nanomolar γ blocks the activation of PDE by micromolar Tα‐GTPγS. The degree of activation of PDE depends reciprocally on the concentrations of γ and Tα‐GTPγS. γ remains bound to the disk membrane during the activation of PDE by transducin. The binding of γ to the αβ subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of Tα‐GTP.
Properties of a second sensory receptor protein inHalobacterium halobiumphototaxis
Authors: Spudich Elena N. , Sundberg Steven A. , Manor Danny , Spudich John L.
Published: May 2005

A second slow‐cycling retinylidene protein, in addition to slow‐cycling (sensory) rhodopsin (SR), can be bleached with hydroxylamine and regenerated with all‐transretinal in photosensory signalingHalobacterium halobiummembranes. Flash photolysis shows this protein undergoes a photochemical reaction cycle characterized by photoconversion of its ground state (λmax480 nm) to a species with λmax⩽ 360 nm, which thermally regenerates the 480‐nm species with a t½of 260 msec at 25°C, under conditions in which SR photocycles at 650 msec in the same membranes. Mutants characterized with respect to their phototaxis behavior are identified which contain SR and the 480‐nm pigment, the latter ranging from undetectable to a concentration equal to that of SR. Receptor mutants lacking all phototaxis sensitivity lack both of the photochemically reactive proteins. The mutant properties contribute to an accumulation of behavioral and spectroscopic evidence that the 480‐nm pigment is a second sensory photoreceptor inH. halobium. NaDodSO4‐polyacrylamide gel electrophoresis of [3H]retinal‐labeled membrane proteins from the mutants indicates SR and the 480‐nm pigment contain distinct chromophoric polypeptides differing in their migration rates. The data implicate polypeptides of 25,000 Mrand 23,000 Mras retinal‐binding polypeptides of SR and the 480‐nm protein, respectively.
Isolation and analysis of arc repressor mutants: Evidence for an unusual mechanism of DNA binding
Authors: Vershon Andrew K. , Bowie James U. , Karplus Theresa M. , Sauer Robert T.
Published: May 2005

We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22. Many of the mutant proteins with substitutions in the C‐terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities. Mutations in the N‐terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three‐dimensional structure. We argue that these N‐terminal residues are important for operator recognition but that they are not part of a conventional helix‐turn‐helix DNA binding structure. These results suggest that Arc may use a new mechanism for sequence specific DNA binding.
Primary structure of a precursor to the asprartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen
Authors: Boel Esper , Bech Anne‐Marie , Randrup Karen , Draeger Bodil , Fiil Neils Peter , Foltmann Bent
Published: May 2005

In order to characterize the zymogen of the milk‐clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase‐specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined.

The decuced amino sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47‐amino‐acid‐long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acide residues with a total molecular weight of 38,701. Clusters of idendtities around the active site cleft support the assumption that these proteinasess have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N‐glycosylation sites were identified.
Multiple conformations of amino acid residues in ribonuclease A
Authors: Svensson L. Anders , Sjölin Lennart , Gilliland Gary L. , Finzel Barry C. , Wlodawer Alexander
Published: May 2005

The highly refined 1.26 Å structure (R = 0.15) of phosphate‐free bovine pancreatic ribonuclease A was modeled with 13 residues having discrete multiple conformations of side chains. These residues are widely distributed over the protein surface, but only one of them, Lys 61, is involved in crystal packing interactions. The discrete conformers have no unusual torsion angles, and their interactions with the solvent and with other atoms of the protein are similar to those residues modeled with a single conformation. For three of the residues–Val 43, Asp 83, and Arg 85–two correlated conformations are found. The observed multiple conformations on the protein surfaces will be of significance in analyzing structure‐function relationships and in performing protein engineering.
Predicted calmodulin‐binding sequence in the γ subunit of phosphorylase b kinase
Authors: Degrado William F. , Erickson‐Viitanen Susan , Wolfe Henry R. , O'Neil Karyn T.
Published: May 2005

A basic, amphiphilic α helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin‐binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin‐binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (γ) subunit of phosphorylase b kinase for interaction with calmodulin (the δ subunit). A peptide corresponding to this site (residues 341–361 of the γ subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the γ subunit) was replaced with an asparagin was found to bind calmodulin with approximately a 3 nM dissociation constant.
Characterization of a slow folding reaction for the α subunit of tryptophan synthase
Authors: Hurle Mark R. , Michelotti Greg A. , Crisanti Mark M. , Matthews C. Robert
Published: May 2005

The equilibria and kinetics of ureainduced unfolding and refolding of the α subunit of tryptophan synthase of E. coli have been examined for their dependences on viscosity, pH, and temperature in order to investigate the properties of one of the rate‐limiting steps, domain association. A viscosity enhancer, 0.58 M sucrose, was found to slow unfolding and accelerate refolding. This apparently anomolous results was shown to be due to the stabilizting effect of sucrose on the folding reaction. After accounting for this stabilization effect by using linear free‐energy plots, the unfolding and refolding kinetics were found to have a viscosity dependence. A decrease in pH was found to stabilize the domain association reaction by increasing the refolding rate and decreasing the unfolding rate. This effect was accounted for by protonation of a single residue with a pK value of 8.8 in the native state and 7.1 in the intermediate, in which the two domains are not yet associated. The activation energy of unfolding is 4.8 kcal/mol, close to the diffusion limit. The negative activation entropy of unfolding, −47 cal/deg‐mol, which controls this reaction, may result from ordering of solvent about the newly exposed domain interface of the transition state. These results may provide information on the types of noncovalent interactions involved in domain association and improve the ability to interpret the folding of mutants with single amino‐acid substitutions at the interface.
Contribution of arginine (HC3) 141α to the Bohr effect of the fourth binding step in the reaction of ligand with human hemoglobin
Authors: Kwiatkowski Laura D. , Noble Robert W.
Published: May 2005

A few years ago we reported that histidine (HC3) 146β plays a major role in the pH‐dependent propeties of the R‐state of human hemoglobin, accounting for close to 50% of the R‐state Bohr effect. We have extended these studies by examining the role of arginine 141α, another group known to affect the overall Bohr effect. We have compared the pH dependencies of the rate constants for the dissociation and combination of the fourth carbon monoxide molecule, I4 and I′4, respectively, for native hemoglobin A (HbA) and a control reconstututed HbA, and des‐(Arg 141α) HbA, the hemoglobin molecule resulting from the enzymatic removal of the C–terminal arginine of the α‐chain of human Hb. From these kinetic contants the pH dependence of L4, by about 80% between pH 6 and 8, where the aldkaline Bohr effect normally occurs, The sum of the effects of the removal of His 146β and of Arg 141α is grater than 100%. This suggests that at least one of these modifications altrs the contrubutions of other residues of this Bohr effect.
Extension of the fragment method to calculate amino acid zwitterion and side chain partition coefficients
Authors: Abraham Donald J. , Leo Albert J.
Published: May 2005

The fragment method of calculation partition coefficients (P) has been extended to include the common amino acids (AAs). The results indicate that polar and charged side chains influence the hydrophobicity of atoms in the side chain in a predictable manner. Feild effects, as evidenced through polar proximity factors and bond factors, need to be considered for accurate estimation of transfer phenomena. The calculated log P and ΔG°′ values of the 20 AAs agree well with the observed values. Pro calculates to be more hydrophilic than the observed log P. Hydrophobicity scales for peptide side chain residues are compared and evaluated in terms of suitability. Calculated π values for nonpolar side chain residures agree well with the observed values; calculated values for uncharged polar side chain residues deviate by about 0.6 log units except for Gln and Cys; and polar side chain residues with charged side chains calculate as too hydorphilic. Reasons for the differences are explored. We also suggest that tightly bound water to polar moieties in amino acids and peptides may be transferred into the octanol phase during partitioning experiments. A quantitative methodology is persented which characterizes the thermodynamic partitioning of groups and individual atoms in amino acids and proteins.
Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase
Authors: Burke J. , Hwang P. , Anderson L. , Lebo R. , Gorin F. , Fletterick R.
Published: May 2005

The intron/exon organization of the human gene for glycogen phosphorylase has been determined. The segments of the polypeptide chain that corresponds to the 19 exons of the gene are examined for relationships between the three‐dimensional structure to the protein and gene structure. Only weak correlations are observed between domains of phosphorylase and exons. The nucleotide binding domains that are found in phosphorylase and other glycolytic enzymes are examined for relationships between exons of the genes and structures of the domains. When mapped to the three‐dimensional structures, the intron/exon boundaries are shown to be widely distributed in this family of protein domains.
Crystallization of purified recombinant human interleukin‐1β
Authors: Carter D. B. , Curry K. A. , Tomich C‐S. C. , Yem A. W. , Deibel M. R. , Tracey D. E. , Paslay J. W. , Carter J. B. , Theriault N. Y. , Harris P. K. W. , Reardon I. M. , Zurcher‐Neely H. A. , Heinrikson R. L. , Clancy L. L. , Muchmore S. W. , Watenpaugh K. D. , Einspahr H. M.
Published: May 2005

The gene for human interleukin‐1β was cloned from SK‐hep‐1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL‐1β) and as a variant with an additional sequence of three amino acids on the N‐terminus (rIL‐1β+). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast‐performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on twodimensional (2‐D) gels as a single spot with a pI of 6.7 ± 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL‐1β have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P41 or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 Å resolution. A structure determination based on these crystals is under way.
Comparison of model and nuclear magnetic resonance structures for the human inflammatory protein C5a
Authors: Zuiderweg Erik R. P. , Henkin Jack , Mollison Karl W. , Carter George W. , Greer Jonathan
Published: May 2005

The model structure previously proposed for human C5a, based upon the crystal structure of the homologous protein human C3a, is compared to the solution structure of human C5a recently determined by nuclear magnetic resonance (NMR) methods in our laboratory. The general folding and helix topography of the C5a protein were modeled very well. The N‐terminus, which is disordered in teh C3a crystal, was correctly predicted in the C5a model both as to its being a helix and as to its docking site on the rest of the molecule. On the other hand, the NMR data show that the biologically important C‐terminal residues are disordered in solution, unlike the model and the C3a crystal structure where this region was helical.