Envelope: interactive software for modeling and fitting complex isotope distributions

Background  

An important aspect of proteomic mass spectrometry involves quantifying and interpreting the isotope distributions arising from mixtures of macromolecules with different isotope labeling patterns. These patterns can be quite complex, in particular with in vivo metabolic labeling experiments producing fractional atomic labeling or fractional residue labeling of peptides or other macromolecules. In general, it can be difficult to distinguish the contributions of species with different labeling patterns to an experimental spectrum and difficult to calculate a theoretical isotope distribution to fit such data. There is a need for interactive and user-friendly software that can calculate and fit the entire isotope distribution of a complex mixture while comparing these calculations with experimental data and extracting the contributions from the differently labeled species.     

An easy-to-use tool for planning and modeling a calorimetric titration

Isothermal titration calorimetry (ITC) is widely employed to measure thermodynamic properties of binding interactions between two macromolecules or a macromolecule and a small ligand. No labeling of interacting species is required for ITC, but this advantage is offset by potentially material-consuming experimental optimization complicated by an indirect readout of an ITC titration. Here we present a simple, practical, and portable spreadsheet-based tool for planning and modeling an ITC titration experiment accompanied by basic guidelines.     

Determination of small differences in buoyant density of macromolecules by analytical density gradient equilibrium centrifugation.

A sensitive method is proposed for the determination of small differences between the buoyant densities of different species of monodisperse macromolecules by analytical density gradient equilibrium centrifugation. The procedure involves the measurement at sedimentation equilibrium of the bandwidths of the concentration distribution of the separate macromolecules and of a mixture of the different species. The difference in buoyant densities can then be estimated from the difference between the bandwidths.     

Determination of Ensemble-Average Pairwise Root Mean-Square Deviation from Experimental B-Factors

Root mean-square deviation (RMSD) after roto-translational least-squares fitting is a measure of global structural similarity of macromolecules used commonly. On the other hand, experimental x-ray B-factors are used frequently to study local structural heterogeneity and dynamics in macromolecules by providing direct information about root mean-square fluctuations (RMSF) that can also be calculated from molecular dynamics simulations. We provide a mathematical derivation showing that, given a set of conservative assumptions, a root mean-square ensemble-average of an all-against-all distribution of pairwise RMSD for a single molecular species, <RMSD²>^1/2, is directly related to average B-factors (<B>) and <RMSF²>^1/2. We show this relationship and explore its limits of validity on a heterogeneous ensemble of structures taken from molecular dynamics simulations of villin headpiece generated using distributed-computing techniques and the Folding@Home cluster. Our results provide a basis for quantifying global structural diversity of macromolecules in crystals directly from x-ray experiments, and we show this on a large set of structures taken from the Protein Data Bank. In particular, we show that the ensemble-average pairwise backbone RMSD for a microscopic ensemble underlying a typical protein x-ray structure is ∼1.1 Å, under the assumption that the principal contribution to experimental B-factors is conformational variability.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Frontal gel chromatography of interacting systems: theoretical and experimental evaluation of the shapes of elution profiles for systems of the type A + B in equilibrium C

A theoretical and experimental study has been made of the advancing elution profile in frontal gel chromatography of interacting systems for which the elution volume of the complex is smaller than that of the larger reactant. First, Gilbert-Jenkins theory is used to delineate the form of the elution profile from the magnitudes of the elution volumes and concentrations of reacting species. This procedure resulted in the detection of a misinterpretation of certain patterns obtained in a gel chromatographic study of the interaction between myoglobin and ovalbumin. Second, a numerical computational procedure, which incorporates both axial dispersion and concentration-dependence of species elution volumes, is used to establish the influence of these two factors on boundary shapes for such systems. Third, frontal gel chromatography on Sephadex G-75 is used to compare experimental behavior with theoretical profiles predicted for the electrostatic interaction between cytochrome c and soybean trypsin inhibitor (pH 6.8, I 0.01). Results of these experiments serve as a guide for future conduct of experiments aimed at characterization of biologically important, reversible complex formation between proteins and/or other macromolecules.     

Reactive nitrogen species in cellular signaling

The transduction of cellular signals occurs through the modification of target molecules. Most of these modifications are transitory, thus the signal transduction pathways can be tightly regulated. Reactive nitrogen species are a group of compounds with different properties and reactivity. Some reactive nitrogen species are highly reactive and their interaction with macromolecules can lead to permanent modifications, which suggested they were lacking the specificity needed to participate in cell signaling events. However, the perception of reactive nitrogen species as oxidizers of macromolecules leading to general oxidative damage has recently evolved. The concept of redox signaling is now well established for a number of reactive oxygen and nitrogen species. In this context, the post-translational modifications introduced by reactive nitrogen species can be very specific and are active participants in signal transduction pathways. This review addresses the role of these oxidative modifications in the regulation of cell signaling events.     

Zone Centrifugation in a Cesium Chloride Density Gradient Caused by Temperature Change

In this communication is described a new technique for the determination of sedimentation coefficients of macromolecules banded in equilibrium density gradients. Initially, the macromolecules are banded in the analytical ultracentrifuge at a low temperature of about 5°C. After equilibrium has been obtained, the temperature is increased to 25°C. The equilibrium band will now sediment to a new equilibrium position in the ultracentrifuge cell: (a) By following the position of the migrating band as a function of time, sedimentation coefficients may be determined. (b) If several species having different sedimentation coefficients are present in the original band, then during the course of the migration the band may split into several new bands which eventually reunite at the final equilibrium position. (c) If different chemical species of macromolecules such as nucleic acids and carbohydrates are present, in general they will exhibit different temperature density relationships, and can move different distances and directions in response to temperature change.     

Structural proteomics of macromolecular assemblies using oxidative footprinting and mass spectrometry

Understanding the composition, structure and dynamics of macromolecules and their assemblies is at the forefront of biological science today. Hydroxyl-radical-mediated protein footprinting using mass spectrometry can define macromolecular structure, macromolecular assembly and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side-chain groups with covalent-modification reagents. Subsequent to oxidation by reactive oxygen species, proteins are digested by specific proteases to generate peptides for analysis by mass spectrometry. Accurate measurements of side-chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side-chain sites within the macromolecular probes are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes.     

The biological essence of resting cells in cell populations

Turnover of cell macromolecules and the diversity of turnover rates in cycling and resting cells is proposed as the underlying fundamental basis for a set of biological phenomena that involve growth, aging, and resistance of normal and tumor cell populations to damage by alkylating agents.It is postulated that in cycling cells the degradation rates are minimal or approaching zero, while in resting cells they are reaching the maximal possible values which are inherent characteristic features of each biological species. The biological advantage of the resting state lies in the ability of these cells to turn to maximal rates of degradation of cell macromolecules. During this process the level of accumulated cellular misinformation infthe form of altered misfunctioning macromolecules is substantially reduced and the cell becomes partially or completely rejuvenated. All cell populations are thought to contain a pool of rejuvenating resting cells, which have a certain probability of reverting to the cycling state.     

Kinetics of electron transfer in macromolecules]

With the help of retarded double-time Green function technique a method of estimation of electron transfer rate in protein macromolecules has been developed. Possible experimental test is suggested.     

Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation

Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c(s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s approximately M(2/3) power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c(s,f(r)), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c(M,R(S)), and sedimentation coefficient-molar mass distributions c(s,M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.     

Organismal Aging and Oxidants beyond Macromolecules Damage

The relationship between oxidants and organismal aging was first articulated through the free radical theory of aging. One of the major predictions of the free radical theory of aging is that oxidative stress shortens organisms’ lifespan because of an increased level of oxidants, which are damaging to macromolecules. However, challenging the role of oxidants in age‐related diseases, there is now sufficient evidence that antioxidant supplements do not provide significant health benefits. Interestingly, in addition to an increase in oxidant‐mediated macromolecules damage, there is convincing experimental data to support the role of senescent cells in the process of aging. Here, the current knowledge regarding the role of oxidants and cellular senescence in organismal aging is reviewed and it is proposed that, in addition to the role of oxidants as inducers of macromolecular damage, oxidants may also function as regulators of signaling pathways involved in the establishment of cellular senescence. If this role for oxidants is established, it may be necessary to modify the free radical theory of aging from “Organisms age because cells accumulate reactive oxygen species‐dependent damage over time” to: “Organisms age because cells accumulate oxidants’‐dependent damage and oxidants’‐dependent senescent characteristics over time.”     

The evaluation of standard sedimentation coefficient in band sedimentation of macromolecules

Standardization in the calculation of the sedimentation coefficient of macromolecules by means of band techniques is discussed. When a sample of macromolecules suspended in a solvent is layered on to a denser bulk solution, the macromolecules do not sediment in this solution alone, but sediment in a mixture of bulk solution and sample solvent. This is caused by the diffusion between sample solvent and bulk solution. Experimental evidence of this process is shown during band sedimentation of ribosomes when the variation of the density and the viscosity along the cell is measured. The calculation shows that in the experimental conditions frequently used, standardization made in the usual way leads to a sedimentation coefficient which is largely overestimated; while standardization yields the correct coefficient if the diffusion effect of the sample solvent in to the bulk solution is taken into account, together with possible deuteration effects. A method to calculate the standard coefficient with the aid of a computer is proposed.     

The ultrasonic degradation of biological macromolecules under conditions of stable cavitation. II. Degradation of deoxyribonucleic acid

Solutions of T7 bacteriophage or calf thymus DNA arc degraded in solution by ultrasonic fields of low intensity in the presence of vibrating air bubbles but are not degraded at these low intensities when such bubbles are absent. Evidence is presented for the hydrodynamic nature of the observed degradation and theoretical simulation of a plausible degradation mechanism is compared with experimental degradation studies. It is concluded that degradation of such linear macromolecules as DNA may occur as a result of stresses induced in the macromolecule; these stresses are the result of a relative movement of solvent molecules and the macromolecules in the time-independent flow of solvent near the vibrating bubbles.     

Mapping of isozymic differences in enolase

The existence of the isozymes of non-regulatory enzymes often has been linked to their interaction with other macromolecules. Enolase, a non-regulatory enzyme, has three isozymes for which sequences have been determined in two or more vertebrate species. The positions in the enolase sequences that differ between the isozymes were mapped in the 3-D structure of the enzyme. The positions in a given isozymic form which were not conserved in different species were considered to be resulting from the neutral drift of sequences and rejected. Also, the residues with no accessible surface were rejected. Three areas with relatively high densities of isozymic substitutions were found. We consider them as the likely sites of contact with other macromolecules.     

Effect of ionic strength on the diffusion coefficient of chondroitin sulfate and heparin measured by quasielastic light scattering

The normal modes for a mixture of charged macromolecules and electrolyte solution are calculated. We derive a generalized Debye relaxation time and the apparent diffusion coefficient of the macroion, which is shown to increase from its Stokes value, obtained in excess of added salt, with decreasing ionic strength. We test our result with experimental data for macromolecules with different charge densities: heparin and chondroitin sulfate. Besides, we show for this latter molecule that while the diffusion coefficient is increased, the scattered intensity is decreased but not by the same factor. Our results are compared with other theories developed in quasielastic light scattering.     

Isolation of in vivo radiolabeled proteoglycans from rat lung

Sulfated macromolecules of rat lung tissue were labeled in vivo with 35SO4 and extracted with a solution of 7 M urea containing 0.4% Triton X-100. DEAE-Sephacel chromatography separated sulfated macromolecules into three pools. Pool I consisted of high-molecular-weight, low-density sulfated glycoprotein, probably of mucous secretion origin. Pool II contained a mixture of proteoheparan sulfate and proteodermatan sulfate, together with core protein-free heparan sulfate chains. Pool III was very heterogeneous; its resolution into at least four proteoglycan species was achieved by CsCl density gradient centrifugation. Those included two (high- and low-density) species of proteoheparan sulfate, high-density proteochondroitin sulfate, and medium-density (1.45 less than rho less than 1.55 g/ml) proteodermatan sulfate.     

The macromolecule with antimicrobial activity synthesized by <Emphasis Type="Italic">Pseudoalteromonas luteoviolacea</Emphasis> strains is an <Emphasis Type="SmallCaps">l</Emphasis>-amino acid oxidase

Two purple pigmented bacterial strains, CPMOR-1 and CPMOR-2, have been newly isolated from the Mediterranean Sea. 16S RNA sequencing and phenotypic characteristics indicate that they belong to the species Pseudoalteromonas luteoviolacea. The synthesis of macromolecules with antimicrobial activity is a capacity described in many strains of this species although the nature of those macromolecules has not been reported up to now. The search for antimicrobial compounds in the two new strains described in this work shows that they synthesize a macromolecule with antimicrobial activity that can be inhibited by catalase, as it had been described in the type strain P. luteoviolacea NCIMB 1893(T). This work elucidates the nature of such macromolecule as a novel L-amino acid oxidase (LAO) with broad substrate specificity. The enzyme is most active with Met, Gln, Leu, Phe, Glu, and Trp. In growth media containing those amino acids, the hydrogen peroxide generated by the reaction catalyzed by the LAO mediates its antimicrobial activity.