DsRed is a recently cloned 28-kDa fluorescent protein responsible
for the red coloration around the oral disk of a coral of the
Discosoma genus. DsRed has attracted tremendous interest
as a potential expression tracer and fusion partner that would be
complementary to the homologous green fluorescent protein from
Aequorea, but very little is known of the biochemistry
of DsRed. We now show that DsRed has a much higher extinction
coefficient and quantum yield than previously reported, plus excellent
resistance to pH extremes and photobleaching. In addition, its 583-nm
emission maximum can be further shifted to 602 nm by mutation of Lys-83
to Met. However, DsRed has major drawbacks, such as strong
oligomerization and slow maturation. Analytical ultracentrifugation
proves DsRed to be an obligate tetramer in vitro, and
fluorescence resonance energy transfer measurements and yeast
two-hybrid assays verify oligomerization in live cells. Also, DsRed
takes days to ripen fully from green to red in vitro or
in vivo, and mutations such as Lys-83 to Arg prevent the
color change. Many potential cell biological applications of DsRed will
require suppression of the tetramerization and acceleration of the
Temporal patterning of biological variables, in the form of
oscillations and rhythms on many time scales, is ubiquitous. Altering
the temporal pattern of an input variable greatly affects the output of
many biological processes. We develop here a conceptual framework for a
quantitative understanding of such pattern dependence, focusing
particularly on nonlinear, saturable, time-dependent processes that
abound in biophysics, biochemistry, and physiology. We show
theoretically that pattern dependence is governed by the nonlinearity
of the input–output transformation as well as its time constant. As a
result, only patterns on certain time scales permit the expression of
pattern dependence, and processes with different time constants can
respond preferentially to different patterns. This has implications for
temporal coding and decoding, and allows differential control of
processes through pattern. We show how pattern dependence can be
quantitatively predicted using only information from steady,
unpatterned input. To apply our ideas, we analyze, in an experimental
example, how muscle contraction depends on the pattern of motorneuron
Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc–sulfur centers as possible targets of selenium redox biochemistry. Selenium compounds release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Aromatic selenium compounds are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidation state +2), benzeneselenenyl chloride (oxidation state 0) and selenocystamine (oxidation state −1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, respectively. Selenium compounds also catalyze the release of zinc from MT in peroxidation and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-butyl hydroperoxide...
A geometric model for the arrangement of phospholipid and protein in biological membrane systems has been proposed. The essential principle underlying this model is that when membrane proteins polymerize, the points of contact between proteins are few, and cavities lined with predominantly nonpolar amino acids are formed. Phospholipid molecules become oriented with the fatty chains inserted into the cavities while the polar heads remain on the surface of the membrane. This orientation applies to both faces of the membrane continuum. All the lipid known to be present in membranes can be accommodated in this manner. The body of evidence supporting this model has been presented.
Experiments on model peptides show that the rate of deamidation of asparaginyl residues depends strongly on the nature of neighboring residues. The natural distribution of glutaminyl and asparaginyl residues is ordered with respect to the biological lifetime of the peptides and the functional groups of the residues neighboring to glutaminyl and asparaginyl residues. The rates of deamidation of such amide peptides under physiological conditions could serve as useful timers of development and aging.
The following question has recently arisen in the literature concerning the interpretation of the optical activity of biological membranes: do the characteristic spectral distortions observed for diverse membrane systems reflect some common and unique aspect of membrane architecture or are they the result of scattering effects owing to the particulate nature of membranous systems? We have confirmed the latter interpretation on the basis of the following experimental observations: (a) red blood cell membranes give a normal circular dichroism spectrum when scattering is reduced and (b) nonaggregated, nonmembranous helical proteins give distorted membranelike spectra when scattering is introduced. An improved estimate of secondary structure on the basis of undistorted spectra results in about 50 per cent α-helix for red blood cell membrane protein. In addition we conclude that the distortions in optical activity spectra offer no evidence in support of various proposed membrane models.
A wide variety of proteins have been shown to bind identical amounts of an amphiphile, sodium dodecyl sulfate, on a gram per gram basis at monomer equilibrium concentrations above 0.5 mM. The binding is independent of ionic strength and primarily hydrophobic in nature. Only the monomeric form of the amphiphile binds to proteins, not the micellar form. The application of these results to models for biological membranes and to gel electrophoresis in sodium dodecyl sulfate is discussed.
A group of peptides (some or all of them glycopeptides), of molecular weight about 5000, has been shown to be a major fraction of biological membranes. These „miniproteins” have been prepared from membranes of human and bovine red blood cells, from purified bovine liver mitochondria, and from the rhodopsin-containing membranes of the outer segments of bovine retinal rods. While the miniproteins were found in large amounts in each case, the other protein components differed markedly depending on the function of the membrane studied. This fact was particularly clear in the case of the rod membranes where the only major proteins detected were the miniproteins and rhodopsin. The large size of the miniprotein fraction in each of these membranes leads us to propose that the miniproteins play a fundamental role in the several functions which occur as common denominators in biological membranes.
This paper describes the synthesis and biological activity of [5-L-asparagine-α,β,β-d3]-deamino-oxytocin. The model peptide S-benzyl-β-mercaptopropionyl-L- asparaginyl-α,β,β-d3-glycinamide was also prepared to observe the effect of the conditions of peptide synthesis on the deuterium content. It was found that normal synthetic procedures could be used without a significant loss in the deuterium content.
Chromous ion reacts with ferricytochrome c to yield a one-to-one Cr(III)-ferrocytochrome c complex. This material, when hydrolyzed by trypsin and subjected to chromatographic procedures, yielded two fragments containing chromium. The amino-acid compositions and chemical characteristics of each of these fragments indicated that the chromium had crosslinked two segments of polypeptide chain; these were residues 40-53-Cr(III)-residues 61-72 and residues 40-53-Cr(III)-residues 61-73. Examination of a model of the ferricytochrome c molecule indicated that only two residues of the crosslinked peptides were sufficiently close to allow crosslinking to take place. These residues were tyrosine 67 and asparagine 52. Enzymatic hydrolysis of one of those fragments by aminopeptidase M supported this identification. The position of the chromic ion implies what is the path of electron transfer from the chromous ion to the ferric ion in this chemical reduction of cytochrome c, and suggests a possible path of electron transfer in biological oxidation-reduction reactions.
The enhancement of DNA synthesis by epidermal growth factor (EGF) in cultured human fibroblasts is demonstrable 24 hr after incubation of the cells at 37°C with very low concentrations (0.83 nM) of the hormone for very short periods (30 min) followed by thorough washing of the cells to remove the free hormone in the medium. This effect must result from persistent, extraordinarily tight binding of the hormone to surface receptors, because the addition of specific, purified anti-EGF IgG as late as 8 hr after initial hormone exposure can completely reverse the biological effects of the hormone. This causes only a slight (but significant) increase in the rate of dissociation at 37°C of the cell-bound 125I-labeled EGF at low occupancy. Together with the fact that in the presence or absence of antibody virtually all of the demonstrable cell-bound 125I-labeled EGF can be shown to dissociate from the cell during a period as short as 2-3 hr, the data suggest the possibility that the biological effects of this hormone may be mediated by occupation of only a negligible fraction of very high affinity binding sites. Thus, the processes of hormone internalization, degradation, and “down regulation” may be irrelevant to the effects of the hormone on DNA synthesis. For this effect the crucial and limiting processes appear to be strictly related to the continuous and persistent occupation of cell surface receptors.
The purification and chemical properties of thymosin, obtained from bovine thymus tissue, are described. The biological activity of the thymic hormone has been assessed by a newly developed rosette assay, which permits measurement of thymus-dependent lymphoid cells. Thymosin activity is associated with a physico-chemically homogeneous protein of molecular weight 12,600. The hormonal activity is evident in in vitro incubation assay, after injection into adult thymectomized mice, and in prolonging survival of neonatally thymectomized mice and the reconstitution of their response to a skin allograft.
Nerve growth factor was insolubilized by covalent attachment to Sepharose beads. Nerve growth factor-Sepharose was biologically active in both the neurite outgrowth assay for nerve growth factor and in preserving responsive neurons in vitro. Modification of the bioassay to detect solubilized activity of nerve growth factor and histological examination of ganglia treated with nerve growth factor-Sepharose revealed that nerve growth factor-Sepharose prepared by reaction in 6 M guanidine hydrochloride released negligible amounts of solubilized nerve growth factor activity. These observations extend the previously noted correlations on the structure and function of nerve growth factor and insulin to include the primary action of these two proteins. Thus nerve growth factor, like insulin, appears to express its biological activity by first binding to a receptor on the surface membrane of responsive cells.
An octacosapeptide with the entire sequence proposed for the newly isolated vasoactive intestinal polypeptide was synthesized and had the expected biological properties. Synthetic peptides corresponding to the C-terminal hendeca-, tetradeca-, pentadeca-, and docosapeptide sequences of the vasoactive intestinal polypeptide showed activities that increased with increasing chain length.
1,25-Dihydroxycholecalciferol, the apparent active hormonal form of cholecalciferol (vitamin D2), is formed from cholecalciferol by specific and sequential hydroxylations of the sterol at carbons 25 and 1. Recently, 1α-hydroxycholecalciferol was synthesized and we report on its biological activity in rachitic chicks. 1α-Hydroxycholecalciferol is identical in potency to 1,25-dihydroxycholecalciferol in stimulation of intestinal calcium absorption; either sterol elicits a near maximal effect at a dose of 0.3-0.6 nmol. The time-course of action of 1α-hydroxycholecalciferol also parallels that of the active metabolite 1,25-dihydroxycholecalciferol with a maximal increase in calcium transport occurring 5-10 hr after administration of sterol to vitamin D-deficient chicks. 6.5 nmol of 1α-hydroxycholecalciferol causes a doubling in calcium absorption in only 2-3 hr, which is the most rapid physiologic response yet detected for a vitamin D-sterol. 1α-Hydroxycholecalciferol is active also in enhancing bone calcium resorption and, like 1,25-dihydroxycholecalciferol, is at least 10 times as active as cholecalciferol in mobilizing bone calcium and raising plasma calcium concentration. It is concluded that 1α-hydroxycholecalciferol represents a synthetic analog of 1...
Structure-function relationships for vitamin D, its metabolites, and analogs are discussed with particular emphasis on the A-ring conformation. It is emphasized that the A-ring of these seco-steroids consists of a pair of rapidly equilibrating chair conformers. As a consequence, different chair conformations produce different orientations of substituent groups in the A-ring. It is proposed that the 1α-hydroxyl of 1α,25-dihydroxyvitamin D3 or its geometric equivalent in analogs must occupy the equatorial, as opposed to the axial, orientation for optimization of biological activity. This proposal is discussed in relation to existing published data on structure-function relationships and the steroid hormone model of action for the biologically active form of vitamin D. This three-dimensional topological model suggests the synthesis of other vitamin D analogs.
All reactions are accelerated by an increase in temperature, but the magnitude of that effect on very slow reactions does not seem to have been fully appreciated. The hydrolysis of polysaccharides, for example, is accelerated 190,000-fold when the temperature is raised from 25 to 100 °C, while the rate of hydrolysis of phosphate monoester dianions increases 10,300,000-fold. Moreover, the slowest reactions tend to be the most heat-sensitive. These tendencies collapse, by as many as five orders of magnitude, the time that would have been required for early chemical evolution in a warm environment. We propose, further, that if the catalytic effect of a “proto-enzyme”—like that of modern enzymes—were mainly enthalpic, then the resulting rate enhancement would have increased automatically as the environment became cooler. Several powerful nonenzymatic catalysts of very slow biological reactions, notably pyridoxal phosphate and the ceric ion, are shown to meet that criterion. Taken together, these findings greatly reduce the time that would have been required for early chemical evolution, countering the view that not enough time has passed for life to have evolved to its present level of complexity.
Gram-negative bacteria contain an unusual outer membrane that prevents the entry of most currently available antibiotics. This membrane contains a complex glycolipid, LPS, on the exterior. It is not understood how such a large molecule, which can contain hundreds of sugars and six fatty acyl chains, is transported across the cell envelope from its site of synthesis in the cytoplasmic membrane to the cell surface. Using a combination of genetics, biochemistry, and structural biology, we characterized residues in the protein that powers LPS transport to gain mechanistic insight into how ATP hydrolysis is coupled to the biological function of the transporter. These tools help us understand how to design antibiotics targeting this essential pathway.
As aulas práticas são fundamentais para o desenvolvimento de habilidades relacionadas ao aprendizado de ciências. A American Society for Biochemistry and Molecular Biology (ASBMB) listou uma série de habilidades que os estudantes devem desenvolver até o final de um programa de Bioquímica e Biologia Molecular, sendo a maioria delas adquirida em aulas práticas. O aprendizado da prática de investigação científica é um dos objetivos das aulas práticas. Esse trabalho propõe a sistematização e avaliação uma sequência didática que contemple o conteúdo de Bioquímica aliado ao desenvolvimento de habilidades de investigação científica. A sequência foi estruturada e aplicada na disciplina Bioquímica Básica (BB280) da Unicamp, nos cursos de Ciências Biológicas diurno e noturno. A sequência foi organizada em atividades alicerçadas em habilidades de investigação científica, tendo como base a teoria de aprendizagem experiencial. As primeiras atividades são mais estruturadas e menos complexas, tornando-se cada vez menos estruturadas e mais complexas, para o desenvolvimento gradativo de habilidades relacionadas ao aprendizado de ciências. Tradicionalmente, as avaliações das aulas práticas da BB280 consistiam em relatórios feitos em grupo e questões na prova teórica sobre o conteúdo prático. Foram desenvolvidos outros instrumentos de avaliação...
This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/ncomms6961; Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to ?maltose by ?-amylase. The detailed structure of the enzyme?s active site yields insights into the mechanism of action of this important class of enzymes.; This work was supported by a grant from the European Research Agency?Industrial Biotechnology Initiative as financed by the national research councils: Biotechnology and Biological Sciences Research Council (grant number BB/L000423) and Agence Fran?aise de l'Environnement et de la Ma?trise de l'Energie (grant number 1201C102). The Danish Council for Strategic Research (grant numbers 12-134923 and 12-134922). The Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT and the European Community?s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N?283570) funded travel to synchrotrons. P.H.W. acknowledges the experimental assistance of Rebecca Gregory and Dr Victor Chechik. L.L.L. acknowledges the experimental assistance of Dorthe Boelskifte and the ESRF and MAXLAB staff for assistance with data collection.