bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface functionalization of thin-film diamond for highly stable and selective biological interfaces
Authors: Stavis Courtney , Clare Tami Lasseter , Butler James E. , Radadia Adarsh D. , Carr Rogan , Zeng Hongjun , King William P. , Carlisle John A. , Aksimentiev Aleksei , Bashir Rashid , Hamers Robert J.
Published: October 2010

Carbon is an extremely versatile family of materials with a wide range of mechanical, optical, and mechanical properties, but many similarities in surface chemistry. As one of the most chemically stable materials known, carbon provides an outstanding platform for the development of highly tunable molecular and biomolecular interfaces. Photochemical grafting of alkenes has emerged as an attractive method for functionalizing surfaces of diamond, but many aspects of the surface chemistry and impact on biological recognition processes remain unexplored. Here we report investigations of the interaction of functionalized diamond surfaces with proteins and biological cells using X-ray photoelectron spectroscopy (XPS), atomic force microscopy, and fluorescence methods. XPS data show that functionalization of diamond with short ethylene glycol oligomers reduces the nonspecific binding of fibrinogen below the detection limit of XPS, estimated as > 97% reduction over H-terminated diamond. Measurements of different forms of diamond with different roughness are used to explore the influence of roughness on nonspecific binding onto H-terminated and ethylene glycol (EG)-terminated surfaces. Finally, we use XPS to characterize the chemical stability of
Escherichia coli
K12 antibodies on the surfaces of diamond and amine-functionalized glass. Our results show that antibody-modified diamond surfaces exhibit increased stability in XPS and that this is accompanied by retention of biological activity in cell-capture measurements. Our results demonstrate that surface chemistry on diamond and other carbon-based materials provides an excellent platform for biomolecular interfaces with high stability and high selectivity.
Marr on Computational-Level Theories
Authors: Shagrir Oron
Published: October 2010

According to Marr, a computational-level theory consists of two elements, thewhatand thewhy. This article highlights the distinct role of the Why element in the computational analysis of vision. Three theses are advanced: (a) that the Why element plays an explanatory role in computational-level theories, (b) that its goal is to explain why the computed function (specified by the What element) is appropriate for a given visual task, and (c) that the explanation consists in showing that the functional relations between the representing cells are similar to the “external” mathematical relations between the entities that these cells represent.
Semantic Holism in Scientific Language
Authors: Andreas Holger
Published: October 2010

Whether meaning is compositional has been a major issue in linguistics and formal philosophy of language for the last 2 decades. Semantic holism is widely and plausibly considered as an objection to the principle of semantic compositionality therein. It comes as a surprise that the holistic peculiarities of scientific language have been rarely addressed in formal accounts so far, given that semantic holism has its roots in the philosophy of science. For this reason, a model-theoretic approach to semantic holism in the language of science is presented here. This approach preserves compositionality to a large extent.
Causal Equations without Ceteris Paribus Clauses
Authors: Gildenhuys Peter
Published: October 2010

Some writers have urged that evolutionary theory produces generalizations that hold only ceteris paribus, that is, provided “everything else is equal.” Others have claimed that all laws in the special sciences, or even all laws in science generally, hold only ceteris paribus. However, if we lack a way to determine when everything else really is equal, hedging generalizations with the phrase “ceteris paribus” renders those generalizations vacuous. In what follows, I propose a solution to this problem for the case of causal equations from classical population genetics. My proviso is generated on the basis of a couple of conceptual maneuvers, “tricks,” which should be usable elsewhere too.
Natural Selection and the Unity of Functional Analyses
Authors: Roszkowski Adela Helena
Published: October 2010

While the question of whether selected-effects accounts of function or causal-role accounts of function provide the ‘true’ functional analysis has given way to a general pluralistic consensus, Philip Kitcher has suggested that different functional accounts allow for unification. I argue that Kitcher's attempt to unify the two functional analyses fails because he adopts the environment-centered perspective on selection as a premise. The premise is undermined by the role niche construction is likely to play in the context of evolution. Moreover, I raise the tentative suggestion that niche construction may threaten the applicability, or at least the relevance, of selected-effects ascriptions.
Hecke fields of analytic families of modular forms
Authors: Hida Haruzo
Published: September 2010

We make finiteness conjectures on the composite of Hecke fields of classical members of a

p
p

-adic analytic family of slope 0 elliptic modular forms in the vertical case (with fixed level varying weight). In the horizontal case (fixed weight varying

p
p

-power level), we prove the corresponding statements.
How Do Antegrade Enemas Work? Colonic Motility in Response to Administration of Normal Saline Solution Into the Proximal Colon
Authors: Gomez Roberto , Mousa Hayat , Liem Olivia , Hayes John , Di Lorenzo Carlo
Published: September 2010

Objective:

The aim of the study was to evaluate the colonic motor response to the administration of normal saline into the proximal colon.

Patients and Methods:

Pediatric patients undergoing colonic manometry received a saline infusion (10–20 mL/kg) in 10 minutes through the central lumen of a catheter placed into the proximal colon. We compared the number of high‐amplitude propagated contractions (HAPCs), motility index, frequency and propagation of other phasic contractions in the 20 minutes before and after normal saline infusion, meal ingestion, and bisacodyl administration.

Results:

Thirteen patients, mean age 9.4 ± 5.8 years, received the saline infusion (3 in the cecum, 8 in the hepatic flexure, and 2 in the transverse colon). In the first 20 minutes after saline infusion, the number of contractions (P = 0.005), distance of propagation of contractions (P = 0.007), frequency of contractions (P = 0.009), and motility index (P = 0.003) were significantly higher compared with baseline. Mean amplitude and number of HAPCs were not significantly different from baseline. Motility parameters after saline and after ingestion of a meal did not differ. All of the measured motility variables significantly increased after bisacodyl stimulation. Bisacodyl increased the motility index and HAPCs more than either saline infusion (P = 0.002) or meal intake (P < 0.001).

Conclusions:

Infusion of saline into the proximal colon is associated with an increase in colonic motility; however, at the volume and rate used in the present study it does not consistently stimulate HAPCs.
Odour-based natal nest recognition in the zebra finch (Taeniopygia guttata), a colony-breeding songbird
Authors: Caspers Barbara A. , Krause E. Tobias
Published: September 2010

Passerine birds have an extensive repertoire of olfactory receptor genes. However, the circumstances in which passerine birds use olfactory signals are poorly understood. The aim of this study is to investigate whether olfactory cues play a role in natal nest recognition in fledged juvenile passerines. The natal nest provides fledglings with a safe place for sleeping and parental food provisioning. There is a particular demand in colony-breeding birds for fledglings to be able to identify their nests because many pairs breed close to each other. Olfactory orientation might thus be of special importance for the fledglings, because they do not have a visual representation of the nest site and its position in the colony when leaving the nest for the first time. We investigated the role of olfaction in nest recognition in zebra finches, which breed in dense colonies of up to 50 pairs. We performed odour preference tests, in which we offered zebra finch fledglings their own natal nest odour versus foreign nest odour. Zebra finch fledglings significantly preferred their own natal nest odour, indicating that fledglings of a colony breeding songbird may use olfactory cues for nest recognition.
Correlates of rediscovery and the detectability of extinction in mammals
Authors: Fisher Diana O. , Blomberg Simon P.
Published: September 2010

Extinction is difficult to detect, even in well-known taxa such as mammals. Species with long gaps in their sighting records, which might be considered possibly extinct, are often rediscovered. We used data on rediscovery rates of missing mammals to test whether extinction from different causes is equally detectable and to find which traits affect the probability of rediscovery. We find that species affected by habitat loss were much more likely to be misclassified as extinct or to remain missing than those affected by introduced predators and diseases, or overkill, unless they had very restricted distributions. We conclude that extinctions owing to habitat loss are most difficult to detect; hence, impacts of habitat loss on extinction have probably been overestimated, especially relative to introduced species. It is most likely that the highest rates of rediscovery will come from searching for species that have gone missing during the 20th century and have relatively large ranges threatened by habitat loss, rather than from additional effort focused on charismatic missing species.
How to lift a model for individual behaviour to the population level?
Authors: Diekmann O. , Metz J. A. J.
Published: September 2010

The quick answer to the title question is: by bookkeeping; introduce as p(opulation)-state a measure telling how the individuals are distributed over their common i(ndividual)-state space, and track how the various i-processes change this measure. Unfortunately, this answer leads to a mathematical theory that is technically complicated as well as immature. Alternatively, one may describe a population in terms of the history of the population birth rate together with the history of any environmental variables affecting i-state changes, reproduction and survival. Thus, a population model leads to delay equations. This delay formulation corresponds to a restriction of the p-dynamics to a forward invariant attracting set, so that no information is lost that is relevant for long-term dynamics. For such equations there exists a well-developed theory. In particular, numerical bifurcation tools work essentially the same as for ordinary differential equations. However, the available tools still need considerable adaptation before they can be practically applied to the dynamic energy budget (DEB) model. For the time being we recommend simplifying the i-dynamics before embarking on a systematic mathematical exploration of the associated p-behaviour. The long-term aim is to extend the tools, with the DEB model as a relevant goal post.
Stylized facts in microalgal growth: interpretation in a dynamic energy budget context
Authors: Lorena António , Marques Gonçalo M. , Kooijman S. A. L. M. , Sousa Tânia
Published: September 2010

A dynamic energy budget (DEB) model for microalgae is proposed. This model deviates from the standard DEB model as it needs more reserves to cope with the variation of assimilation pathways, requiring a different approach to growth based on the synthesizing unit (SU) theory for multiple substrates. It is shown that the model is able to accurately predict experimental data in constant and light-varying conditions with most of the parameter values taken directly from the literature. Also, model simulations are shown to be consistent with stylized facts (SFs) concerning N∶C ratio. These SFs are reinterpreted and the general conclusion is that all forcing variables (dilution rate, temperature and irradiance) impose changes in the nitrogen or carbon limitation status of the population, and consequently on reserve densities. Model predictions are also evaluated in comparison with SFs on chlorophyll concentration. It is proposed that an extra structure, more dependent on the nitrogen reserve, is required to accurately model chlorophyll dynamics. Finally, SFs concerning extracellular polymeric substances (EPSs) production by benthic diatoms are collected and interpreted and a formulation based on product synthesis and rejection flux is proposed for the EPSs production rate.
Evidence for a common gene pool and frequent recombinational exchange of the tbpBA operon in Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi
Authors: Lee Inkyoung , Davies Robert L.
Published: October 2010

The tbpBA operon was sequenced in 42 representative isolates of Mannheimia haemolytica (32), Mannheimia glucosida (6) and Bibersteinia trehalosi (4). A total of 27 tbpB and 20 tbpA alleles were identified whilst the tbpBA operon was represented by 28 unique alleles that could be assigned to seven classes. There were 1566 (34.8 % variation) polymorphic nucleotide sites and 482 (32.1 % variation) variable inferred amino acid positions among the 42 tbpBA sequences. The tbpBA operons of serotype A2 M. haemolytica isolates are, with one exception, substantially more diverse than those of the other M. haemolytica serotypes and most likely have a different ancestral origin. The tbpBA phylogeny has been severely disrupted by numerous small- and large-scale intragenic recombination events. In addition, assortative (entire gene) recombination events, involving either the entire tbpBA operon or the individual tbpB and tbpA genes, have played a major role in shaping tbpBA structure and it's distribution in the three species. Our findings indicate that a common gene pool exists for tbpBA in M. haemolytica, M. glucosida and B. trehalosi. In particular, B. trehalosi, M. glucosida and ovine M. haemolytica isolates share a large portion of the tbpA gene, and this probably reflects selection for a conserved TbpA protein that provides effective iron uptake in sheep. Bovine and ovine serotype A2 lineages have very different tbpBA alleles. Bovine-like tbpBA alleles have been partially, or completely, replaced by ovine-like tbpBA alleles in ovine serotype A2 isolates, suggesting that different transferrin receptors are required by serotype A2 isolates for optimum iron uptake in cattle and sheep. Conversely, the tbpBA alleles of bovine-pathogenic serotype A1 and A6 isolates are very similar to those of closely related ovine isolates, suggesting a recent and common evolutionary origin.
Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development
Authors: Singh Vinayak , Chandra Deepak , Srivastava Brahm S. , Srivastava Ranjana
Published: October 2010

Acetohydroxyacid synthase (AHAS) is a biosynthetic enzyme essential forde novosynthesis of branched-chain amino acids. The genome sequence ofMycobacterium tuberculosisrevealed genes encoding four catalytic subunits,ilvB1(Rv3003c),ilvB2(Rv3470c),ilvG(Rv1820) andilvX(Rv3509c), and one regulatory subunit,ilvN(Rv3002c), of AHAS. All these genes were found to be expressed inM. tuberculosisgrowingin vitro. Each AHAS subunit gene was cloned and expressed inEscherichia coli. AHAS activity of IlvB1 and IlvG was found in cell-free lysates and with recombinant purified proteins. Kinetic studies with purified IlvG revealed positive cooperativity towards substrate and cofactors. To understand the role of the catalytic subunits in the biology ofM. tuberculosis, expression of AHAS genes was analysed in different physiological conditions.ilvB1,ilvB2andilvGwere differentially expressed. The role ofilvB1in persistence is known, but the upregulation ofilvB2andilvGin extended stationary phase,ex vivo, and in acid stress and hypoxic environments, suggests the relevance of AHAS enzymes in the metabolism and survival ofM. tuberculosisby functioning as catabolic AHAS. These enzymes are therefore potential targets for drug development.
Presence of Salmonella pathogenicity island 2 genes in seafood-associated Salmonella serovars and the role of the sseC gene in survival of Salmonella enterica serovar Weltevreden in epithelial cells
Authors: Bhowmick Patit P. , Devegowda Devananda , Ruwandeepika H. A. Darshanee , Karunasagar Iddya , Karunasagar Indrani
Published: October 2010

The type III secretion system encoded by theSalmonellapathogenicity island 2 (SPI-2) has a central role in the pathogenesis of systemic infections bySalmonella. Sixteen genes (ssaU,ssaB,ssaR,ssaQ,ssaO,ssaS,ssaP,ssaT,sscB,sseF,sseG,sseE,sseD,sseC,ssaDandsscA) of SPI-2 were targeted for PCR amplification in 57 seafood-associated serovars ofSalmonella. ThesseCgene of SPI-2 was found to be absent in two isolates ofSalmonella entericaserovar Weltevreden, SW13 and SW39. Absence ofsseCwas confirmed by sequencing using flanking primers. SW13 had only 66 bp sequence of thesseCgene and SW39 had 58 bp sequence of this gene. A clinical isolate,S. Weltevreden – SW3, 10 : r : z6 – was used to construct a deletion mutant for thesseCgene. Significant reduction in the survival of SW3, 10 : r : z6 ΔsseCand natural mutants SW13 and SW39 in HeLa cells suggests thatsseChas a crucial role in the intracellular survival ofS. Weltevreden. Expression ofsseCwas upregulated during the intracellular phase of bothS. entericaserovar Typhimurium and clinical isolateS. Weltevreden SW3, 10 : r : z6, suggesting a crucial role for this gene in the survival ofS. Weltevreden inside host cells.
Current views on the function of the lymphatic vasculature in health and disease
Authors: Wang Yingdi , Oliver Guillermo
Published: October 2010

The lymphatic vascular system is essential for lipid absorption, fluid homeostasis, and immune surveillance. Until recently, lymphatic vessel dysfunction had been associated with symptomatic pathologic conditions such as lymphedema. Work in the last few years had led to a better understanding of the functional roles of this vascular system in health and disease. Furthermore, recent work has also unraveled additional functional roles of the lymphatic vasculature in fat metabolism, obesity, inflammation, and the regulation of salt storage in hypertension. In this review, we summarize the functional roles of the lymphatic vasculature in health and disease.
TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia
Authors: Mukhopadhyay Saikat , Wen Xiaohui , Chih Ben , Nelson Christopher D. , Lane William S. , Scales Suzie J. , Jackson Peter K.
Published: October 2010

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3–IFT-A interaction suggests how these proteins cooperate during neural tube patterning.
Imaging Articular Cartilage Tissue Using Atomic Force Microscopy (AFM): Figure 1.
Authors: Plodinec Marija , Loparic Marko , Aebi Ueli
Published: October 2010

INTRODUCTION

Cartilage is a complex avascular tissue composed of cells (“chondrocytes”) embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends. Atomic force microscopy (AFM) can be used to examine structure-function relationships of cartilage at both micrometer and nanometer scales. When imaged at the micrometer scale with microspheres, only the ECM and chondrocytes can be distinguished. Correspondingly, mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety. In this protocol, the corresponding AFM image and force map are used to reveal the distinct morphology of the collagen fibers and proteoglycan gel. Although, in principle, these experiments can be performed using any AFM, an AFM with tube scanners that have manual screws for tilting the sample is preferable because cartilage has macroscopically rough surface features. By manually tilting the probe over the sample, an optimal angle for tip approach can be achieved.
Visualizing Axons in the Drosophila Central Nervous System Using Immunohistochemistry and Immunofluorescence: Figure 1.
Authors: Bashaw Greg J.
Published: October 2010

INTRODUCTION

How axons in the developing nervous system navigate to their correct targets is a fundamental question in neuroscience. Studies of axon guidance in the embryonic central nervous system (CNS) of Drosophila melanogaster have proved instrumental to the identification of molecules and mechanisms that regulate wiring. The relative simplicity of the Drosophila embryonic CNS, the advantages of genetic approaches, and the ability to analyze the pathfinding decisions of well-defined axon groups or even individual axons have all contributed to our understanding of the fundamental mechanisms of axon guidance and target selection. In this protocol, we describe methods for using fluorescent antibody staining as well as horseradish peroxidase (HRP) immunohistochemistry to reveal axon-projection patterns and identify specific guidance molecules. In the presence of 3,3′-diaminobenzidine tetrahydrochloride (DAB), HRP forms a brown, stable precipitate that allows visualization of axon populations under white light. This can be quite advantageous when teaching basic embryonic anatomy and dissection techniques, because unlike fluorescent antibody staining, HRP can be visualized in a lighted room. HRP detection does not allow precise colocalization of signals, nor can multiple colors be visualized simultaneously. These objectives are easily achieved with fluorescent antibody staining.
Live Dissection and Surface Labeling of Proteins in Drosophila Embryos
Authors: Bashaw Greg J.
Published: October 2010

INTRODUCTION

This protocol presents methods for the specific detection of neuronal cell-surface receptors and secreted factors. These techniques allow visualization of surface pools of receptor proteins and also reveal the surface distribution of secreted cues. Live dissection of embryos to expose the ventral nerve cord is also described. This procedure can be used to improve the ability to detect signals from suboptimal antibodies, where conventional staining gives poor results.
Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid
Authors: Messing Simon A.J. , Gabelli Sandra B. , Echeverria Ignacia , Vogel Jonathan T. , Guan Jiahn Chou , Tan Bao Cai , Klee Harry J. , McCarty Donald R. , Amzel L. Mario
Published: October 2010

The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-Å resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.
Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin
Authors: Min Tongpil , Ergenekan Can E. , Eidsness Marly K. , Ichiye Toshiko , Kang Chulhee
Published: October 2010

Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 Å resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe—S] cluster and concomitant contraction of the NH • • • S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high‐resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.
Cleavage of the iron‐methionine bond in c‐type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex
Authors: Geremia Silvano , Garau Gianpiero , Vaccari Lisa , Sgarra Riccardo , Viezzoli Maria Silvia , Calligaris Mario , Randaccio Lucio
Published: October 2010

The three‐dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Å and in the reduced state at 1.95 Å resolution. These are the first high‐resolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six‐residue insertion Gly82‐Ala87, which precedes the heme binding Met93, forms an isolated 310‐helix secondary structural element not previously observed in other c‐type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c‐type cytochromes has revealed that the presence of such a residue, with torsion angles ϕ and ξ of approximately −140 and −130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Å resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X‐ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high‐pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c‐type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H‐bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c2 of the rare six‐residue insertion in the helix 310 conformation that increases Met loop flexibility.
From discrete protein kinetics to continuous Brownian dynamics: A new perspective
Authors: Qian Hong
Published: October 2010

This article presents a comparative analysis of two sets of data from recent experiments on kinetics of (i) protein unfolding by mechanical force and (ii) channel gating with membrane electric potential. Both situations necessitate a continuous Brownian‐dynamic view of protein conformational kinetics. We show that the discrete approach traditional to biochemical kinetics is insufficient for understanding dynamics of protein molecules in an aqueous solution or lipid membrane with varying conditions under which the major activation barrier can disappear. A semiquantitative analysis based on Brownian dynamics in a continuous energy landscape offers a more comprehensive description for motions of biological macromolecules.
N‐terminal contributions of the γ‐subunit of fetal hemoglobin to its tetramer strength: Remote effects at subunit contacts
Authors: Yagami Takeshi , Ballard Barry T. , Padovan Julio Cesar , Chait Brian T. , Popowicz Anthony M. , Manning James M.
Published: October 2010

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70‐fold smaller tetramer‐dimer dissociation constant. This property has been shown previously to be only partially caused by the 5‐amino‐acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18‐amino‐acid N‐terminal A helix of the γ‐subunit of fetal hemoglobin, which differs from the β‐subunit of adult hemoglobin at eight amino acid residues. This long‐distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the γ‐subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the β‐subunit by the corresponding residues in the γ‐subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (γ) of HbF with its high pKa of 8.1 compared with a 7.1 value for Val 1 (β) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the γ‐subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (β) by Glu 5 (γ) promotes tetramer strengthening. The Glu → Asp replacement at position 7 enhances this effect because of the lower pKa of Asp but the Val → Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen‐binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer‐dimer allosteric interface, oxygen‐binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.
Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 Å resolution
Authors: Chatani Eri , Hayashi Rikimaru , Moriyama Hideaki , Ueki Tatzuo
Published: October 2010

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild‐type RNase A and three mutants—F120A, F120G, and F120W—were analyzed up to a 1.4 Å resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild‐type RNase A, except for the C‐terminal region of F120G with a high B‐factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild‐type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild‐type RNase A, and those of F120W and F120G appeared to be “inside out” compared with that of wild‐type RNase A. Only ∼1 Å change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.
A cavity‐forming mutation in insulin induces segmental unfolding of a surrounding α‐helix
Authors: Xu Bin , Hua Qing‐Xin , Nakagawa Satoe H. , Jia Wenhua , Chu Ying‐Chi , Katsoyannis Panayotis G. , Weiss Michael A.
Published: October 2010

To investigate the cooperativity of insulin's structure, a cavity‐forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, IleA2→Ala in the A1–A8 α‐helix, does not impair disulfide pairing between chains. In accord with past studies of cavity‐forming mutations in globular proteins, a decrement was observed in thermodynamic stability (ΔΔGu 0.4–1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated α‐helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1–A8 segment. The analog's solution structure is otherwise similar to that of native insulin, including the B chain's supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity‐forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of IleA2, a residue of low helical propensity, stabilizes the A1–A8 α‐helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The AlaA2 analog's low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1–A8 α‐helix in receptor recognition.
Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies
Authors: Yuan Tao , Vogel Hans J.
Published: October 2010

Calmodulin (CaM) is a 148‐residue regulatory calcium‐binding protein that activates a wide range of target proteins and enzymes. Calcium‐saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two “active sites” of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium‐dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM‐binding domain of myosin light‐chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild‐type CaM (WT‐CaM). One‐and two‐dimensional NMR studies of the Eth‐CaM and Nle‐CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth‐CaM and Nle‐CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the KD for Nle‐CaM also increased 3.5‐fold compared with WT‐CaM. These data show that the “active site” Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site‐directed mutagenesis studies of the Met residues of CaM.
KM+, a mannose‐binding lectin from artocarpus integrifolia: Amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the β‐prism fold
Authors: Rosa José César , Greene Lewis Joel , De Oliveira Paulo SÉRgio Lopes , Garratt Richard , Beltramini Leila , Resing Katheryn , Roque‐Barreira Maria‐Cristina
Published: October 2010

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl‐blocked N‐terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine‐rich linker that unites the regions homologous to the α‐and β‐chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (α‐D‐mannose, α‐D‐glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the β‐prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the β‐prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a β‐hairpin containing two classic‐type β‐bulges. We suggest the term β‐prism motif to describe this feature.
Relationships within the aldehyde dehydrogenase extended family
Authors: Perozich John , Nicholas Hugh , Wang Bi‐Cheng , Lindahl Ronald , Hempel John
Published: October 2010

One hundred‐forty‐five full‐length aldehyde dehydrogenase‐related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E‐NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the “Class 3” trunk, contains mostly substrate‐specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the “Class 102” trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate‐specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.
Thermodynamic linkage between the binding of protons and inhibitors to HIV‐1 protease
Authors: Trylska Joanna , Antosiewicz Jan , Geller Maciej , Hodge C. Nicholas , Klabe Ronald M. , Head Martha S. , Gilson Michael K.
Published: October 2010

The aspartyl dyad of free HIV‐1 protease has apparent pKas of ∼3 and ∼6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI‐272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson‐Boltzmann model of electrostatics is used to compute the apparent pKas of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns ϵ = 4 to the solute interior and uses a detailed model of ionization; the other uses ϵ = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pKas of ∼3 and ∼6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pKas in complex protein environments.

Because binding of a cyclic urea inhibitor shifts the pKas of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.
Folding simulation of protein models on the structure‐based cubo‐octahedral lattice with the Contact Interactions algorithm
Authors: Toma Lucio , Toma Salvatore
Published: October 2010

Computer simulations of protein models on lattices have been widely used as an aid in the study of protein folding process. Following the suggestion of Raghunathan and Jernigan (1997, Protein Sci 6:2072—2083) that the cubooctahedral lattice can allow a more realistic representation of proteins than other lattices, we propose here the use of a new set of internal coordinates θ for the description of a protein model on this lattice. An easy procedure for the conversion of the θ coordinates to the Cartesian coordinates is also described. When the Contact Interaction approach, already proposed by us for simulations on square or cubic lattices, was applied to the cubo‐octahedral lattice, the system obeyed the correct thermodynamics derived from the definition of energy. Thus, lattice simulations of protein models, in which secondary structure elements such as α‐helices or β‐strands can be easily identifiable, can be performed.
A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions
Authors: Kaushik Jai K. , Bhat Rajiv
Published: October 2010

The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in‐depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico‐chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 °C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, ΔCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico‐chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.