Synthesis of 3,3'-bis(sulfonato)-4,4'-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

This protocol describes a procedure for the synthesis of 3,3'-bis(sulfonato)-4,4'-bis(chloroacetamido)azobenzene (BSBCA), a water-soluble, thiol-reactive, photo-switchable cross-linker. In addition, a protocol is outlined for installing the cross-linker in an intramolecular fashion onto proteins bearing two surface-exposed Cys residues. BSBCA is designed to be used as an in vitro activity switch that operates by exerting temporal and reversible photo-control over alpha-helix content within synthetic peptides and recombinant proteins. Synthesis of the cross-linker requires approximately 4.5 d, and cross-linking can be performed in 10-12 h.     

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins

Green fluorescent protein and its variants are frequently used as F?rster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (相似文献   

Evidence for a sliding-resistance at the tip of the trypanosome flagellum

Motility in trypanosomes is achieved through the undulating behaviour of a single "9 + 2" flagellum; normally the flagellar waves begin at the flagellar tip and propagate towards the base. For flagella in general, however, propagation is from base-to-tip and it is believed that bend formation, and sustained regular oscillation, depend upon a localised resistance to inter-doublet sliding - which is normally conferred by structures at the flagellar base, typically the basal body. We therefore predicted that in trypanosomes there must be a resistive structure at the flagellar tip. Electron micrographs of Crithidia deanei, Herpetomonas megaseliae, Trypanosoma brucei and Leishmania major have confirmed that such attachments are present. Thus, it can be assumed that in trypanosomes microtubule sliding at the flagellar tip is resisted sufficiently to permit bend formation.     

Non-native interactions play an effective role in protein folding dynamics

Systematic Monte Carlo simulations of simple lattice models show that the final stage of protein folding is an ordered process where native contacts get locked (i.e., the residues come into contact and remain in contact for the duration of the folding process) in a well‐defined order. The detailed study of the folding dynamics of protein‐like sequences designed as to exhibit different contact energy distributions, as well as different degrees of sequence optimization (i.e., participation of non‐native interactions in the folding process), reveals significant differences in the corresponding locking scenarios—the collection of native contacts and their average locking times, which are largely ascribable to the dynamics of non‐native contacts. Furthermore, strong evidence for a positive role played by non‐native contacts at an early folding stage was also found. Interestingly, for topologically simple target structures, a positive interplay between native and non‐native contacts is observed also toward the end of the folding process, suggesting that non‐native contacts may indeed affect the overall folding process. For target models exhibiting clear two‐state kinetics, the relation between the nucleation mechanism of folding and the locking scenario is investigated. Our results suggest that the stabilization of the folding transition state can be achieved through the establishment of a very small network of native contacts that are the first to lock during the folding process.     

Developmental experience alters information coding in auditory midbrain and forebrain neurons

In songbirds, species identity and developmental experience shape vocal behavior and behavioral responses to vocalizations. The interaction of species identity and developmental experience may also shape the coding properties of sensory neurons. We tested whether responses of auditory midbrain and forebrain neurons to songs differed between species and between groups of conspecific birds with different developmental exposure to song. We also compared responses of individual neurons to conspecific and heterospecific songs. Zebra and Bengalese finches that were raised and tutored by conspecific birds, and zebra finches that were cross‐tutored by Bengalese finches were studied. Single‐unit responses to zebra and Bengalese finch songs were recorded and analyzed by calculating mutual information (MI), response reliability, mean spike rate, fluctuations in time‐varying spike rate, distributions of time‐varying spike rates, and neural discrimination of individual songs. MI quantifies a response's capacity to encode information about a stimulus. In midbrain and forebrain neurons, MI was significantly higher in normal zebra finch neurons than in Bengalese finch and cross‐tutored zebra finch neurons, but not between Bengalese finch and cross‐tutored zebra finch neurons. Information rate differences were largely due to spike rate differences. MI did not differ between responses to conspecific and heterospecific songs. Therefore, neurons from normal zebra finches encoded more information about songs than did neurons from other birds, but conspecific and heterospecific songs were encoded equally. Neural discrimination of songs and MI were highly correlated. Results demonstrate that developmental exposure to vocalizations shapes the information coding properties of songbird auditory neurons. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 235–252, 2010.     

Statistical modeling of biomedical corpora: mining the Caenorhabditis Genetic Center Bibliography for genes related to life span

Background  

The statistical modeling of biomedical corpora could yield integrated, coarse-to-fine views of biological phenomena that complement discoveries made from analysis of molecular sequence and profiling data. Here, the potential of such modeling is demonstrated by examining the 5,225 free-text items in the Caenorhabditis Genetic Center (CGC) Bibliography using techniques from statistical information retrieval. Items in the CGC biomedical text corpus were modeled using the Latent Dirichlet Allocation (LDA) model. LDA is a hierarchical Bayesian model which represents a document as a random mixture over latent topics; each topic is characterized by a distribution over words.     

The shape of human gene family phylogenies

Background  

The shape of phylogenetic trees has been used to make inferences about the evolutionary process by comparing the shapes of actual phylogenies with those expected under simple models of the speciation process. Previous studies have focused on speciation events, but gene duplication is another lineage splitting event, analogous to speciation, and gene loss or deletion is analogous to extinction. Measures of the shape of gene family phylogenies can thus be used to investigate the processes of gene duplication and loss. We make the first systematic attempt to use tree shape to study gene duplication using human gene phylogenies.     

Flagellar oscillation: a commentary on proposed mechanisms

Eukaryotic flagella and cilia have a remarkably uniform internal ‘engine’ known as the ‘9+2’ axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive ‘switching’ event is assumed to occur; there are several proposals regarding the nature of the ‘switch’ and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The ‘provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force‐generating cycles and dynein‐to‐dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self‐triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem.