17,055 research outputs found
Water dynamics and stability of major blood proteins at pre-denaturation stage
We investigate the temperature effect on the size and stability of two major blood plasma proteins, human serum albumin and fibrinogen in aqueous NaCl solution. Dynamic Light Scattering measurements were carried out in the physiological temperature range up to 45 degrees C. The analysis of the results provided the temperature dependences of the macromolecular hydrodynamic radius and the potential. For albumin the hydrodynamic radius remained unchanged, while the zeta-potential increased sharply at approximately 40 degrees C. For fibrinogen the radius increased significantly above 45 degrees C and the zeta-potential increased similar to albumin at slightly below 40 degrees C. The dynamics of albumin macromolecule was simulated using classical Molecular Dynamics, which showed no change in the gyration radius, root mean square deviation, and the composition of disulfide and salt bridges, but substantial change in the secondary structure of the protein. We conclude that these changes in the structure and dynamics of the proteins are correlated with the qualitative change of water dynamics at 42 degrees C in the hydration shell of the proteins
The effect of water dynamics on conformation changes of albumin in pre-denaturation state:photon correlation spectroscopy and simulation
Water is essential for protein three-dimensional structure, conformational dynamics, and activity. Human serum albumin (HSA) is one of major blood plasma proteins, and its functioning is fundamentally determined by the dynamics of surrounding water. The goal of this study is to link the conformational dynamics of albumin to the thermal motions in water taking place in the physiological temperature range. We report the results of photon correlation spectroscopy and molecular dynamics simulations of HSA in aqueous solution. The experimental data processing produced the temperature dependence of the HSA hydrodynamic radius and its zeta potential. Molecular dynamics reproduced the results of experiments and revealed changes in the secondary structure caused by the destruction of hydrogen bonds in the macromolecule's globule
Mechanistic behaviour and molecular interactions of heat shock protein 47 (HSP47)
This project involves the study of heat shock protein 47 (HSP47), which is a molecular chaperone crucial for collagen biosynthesis. It exhibits a high degree of sequence homology with members of the serine protease inhibitor (serpin) superfamily, though HSP47 does not possess the inhibitory activity. It is a single-substrate chaperone, and binds only to collagen. ‘Knock-out’ of the hsp47 gene impairs the secretion of correctly folded collagen triple helix molecules leading to embryonic lethality in mice. Thus the aim of this project was to elucidate the specific mechanism that governs the binding to and release from collagen at the molecular level, known as the ‘pH-switch mechanism’. Emphasis is given on histidine (His) residues as the HSP47-collagen dissociation pH is similar to the pKa of the imidazole side chain of His residues. Site directed mutagenesis was used to mutate surface His residues, based on a mouse HSP47 homology model. The effects of the mutations on the behaviour of HSP47 were then assessed by collagen binding assays and structural analyses with circular dichroism (CD). All mutants were found to have good solubility and retain their binding ability to collagen like wild-type HSP47 in batch assay, but perturbed behaviour was seen in column experiment. Mutation of His residue at position 191 (H191) causes the shift in the collagen dissociation pH, while mutation of H197 and/or 198 disrupt the specific HSP47-collagen interaction. H191, 197 and 198 are predicted to be located in the region near the C-terminus of strand 3 of β-sheet A (s3A) in the homology model, a region specifically known as the ‘breach cluster’ in serpin nomenclature. The extent of conformational rearrangement of this region was further investigated by means of intrinsic tryptophan fluorescence spectroscopy using a series of single tryptophan (Trp) mutants. Results from analyses performed on the mutants did not contradict the observation seen in His mutational work, as Trp residues in the ‘breach’ cluster are likely to be located in the dynamic region of HSP47 pH-triggered conformational change. In conclusion, this study establishes the importance of His residues in the ‘breach cluster’ to HSP47 pH-switch behaviour. Finally, a model for HSP47 pH-switch mechanism was proposed from data obtained via mutagenesis experiments. The model is hoped to assist future research into HSP47 cellular behaviour and will also be of great use in therapeutic applications involving the molecular chaperone
Proteins in saccharides matrices and the trehalose peculiarity: Biochemical and biophysical properties
Immobilization of proteins and other biomolecules in saccharide matrices leads to a series of peculiar properties that are relevant from the point of view of both biochemistry and biophysics, and have important implications on related fields such as food industry, pharmaceutics, and medicine. In the last years, the properties of biomolecules embedded into glassy matrices and/or highly concentrated solutions of saccharides have been thoroughly investigated, at the molecular level, through in vivo, in vitro, and in silico studies. These systems show an outstanding ability to protect biostructures against stress conditions; various mechanisms appear to be at the basis of such bioprotection, that in the case of some sugars (in particular trehalose) is peculiarly effective. Here we review recent results obtained in our and other laboratories on ternary protein- sugar-water systems that have been typically studied in wide ranges of water content and temperature. Data from a large set of complementary experimental techniques provide a consistent description of structural, dynamical and functional properties of these systems, from atomistic to thermodynamic level. In the emerging picture, the stabilizing effect induced on the encapsulated systems might be attributed to a strong biomolecule-matrix coupling, mediated by extended hydrogen-bond networks, whose specific properties are determined by the saccharide composition and structure, and depend on water content
Probing Plasmodium falciparum sexual commitment at the single-cell level
Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission. Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells. Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions. Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign
A transient syntaxin/SNAP-25 interaction serves as ready-available binding site for synaptobrevin
Characterization of the longitudinal HIV-1 quasispecies evolution in HIV-1 infected individuals co-infected with Mycobacterium tuberculosis
One of the earliest and most striking observations made about HIV is the extensive genetic variation that the virus has within individual hosts, particularly in the hypervariable regions of the env gene which is divided into 5 variable regions (V1-V5) and 5 more constant (C1-C5) regions. HIV evolves at any time over the course of an individual’s infection and infected individuals harbours a population of genetically related but non-identical viruses that are under constant change and ready to adapt to changes in their environment. These genetically heterogeneous populations of closely related genomes are called quasispecies [65]. Tuberculosis or tubercle forming disease is an acute and/or chronic bacterial infection that primarily attacks the lungs, but which may also affect the kidneys, bones, lymph nodes, and brain. The disease is caused by Mycobacterium tuberculosis (MTB), a slow growing rod-shaped, acid fast bacterium. It is transmitted from person to person through inhalation of bacteria-carrying air droplets. Worldwide, one person out of three is infected with Mycobacterium tuberculosis – two billion people in total. TB currently holds the seventh place in the global ranking of causes of death [73]. In 2008, there were an estimated 9.4 (range, 8.9–9.9 million) million incident cases (equivalent to 139 cases per 100 000 population) of TB globally [75]. A complex biological interplay occurs between M. tuberculosis and HIV in coinfected host that results in the worsening of both pathologies. HIV promotes progression of M. tuberculosis either by endogenous reactivation or exogenous reinfection [77, 78] and, the course of HIV-1 infection is accelerated subsequent to the development of TB [80]. Active TB is associated with an increase in intra-patient HIV-1 diversity both systemically and at the infected lung sites [64,122]. The sustainability or reversal of the HIV-1 quasispecies heterogeneity after TB treatment is not known. Tetanus toxoid vaccinated HIV-1 infected patients developed a transient increase in HIV-1 heterogeneity which was reversed after few weeks [121]. Emergence of a heterogeneous HIV-1 population within a patient may be one of the mechanisms to escape strong immune or drug pressure [65,128]. The existence of better fitting and/or immune escape HIV-variants can lead to an increase in HIV-1 replication [129,130]. It might be that TB favourably selected HIV-1 variants which are sources for consistent HIV-1 replication. Understanding the mechanisms underlying the impacts of TB on HIV-1 is essential for the development of effective measures to reduce TB related morbidity and mortality in HIV-1 infected individuals. In the present study we studied whether the increase in HIV-1 quasispecies diversity during active TB is reversed or preserved throughout the course of antituberculous chemotherapy. For this purpose Two time point HIV-1 quasispecies were evaluated by comparing HIV-1 infected patients with active tuberculosis (HIV-1/TB) and HIV-1 infected patients without tuberculosis (HIV-1/non TB). Plasma samples were obtained from the Frankfurt HIV cohort and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty five clones were sequenced from each patient. Various phylogenetic analyses were performed including tree inferences, intra-patient viral diversity and divergence, selective pressure, co-receptor usage prediction and two time point identity of quasispecies comparison using Mantel’s test. We found out from this study that: 1) Active TB sustains HIV-1 quasispecies diversity for longer period 2. Active TB increases the rate of HIV-1 divergence 3) TB might slow down evolution of X4 variants And we concluded that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV- 1 may be predominant for R5 viruses. The use of CCR5-coreceptor inhibitors for HIV-1/TB patients as therapeutic approach needs further investigation.Eine der ersten und überraschenden Beobachtungen, welche bei der Analyse des HI-Virus gemacht wurden ist seine ausgeprägte Genetische Variabilität besonders die hypervariable Region des env Genes betreffen. Dieses wird in 5 variable Regionen (V1-V5) sowie 5 stärker konservierte Regionen (C1-C5) unterteilt. HIV wandelt sich zu jedem Zeitpunkt im Verlauf der Infektion und jedes infizierte Individuum ist Träger einer Population von genetisch verwandten jedoch nicht identischen Viren, welche sich kontinuierlich verändern und an die Erfordernisse innerhalb der Umgebung anpassen. Diese genetisch heterogenen, jedoch eng verwandten Populationen werden Quasispecies genannt. Tuberkulose ist eine mykobakterielle Infektion, welche sowohl akute als auch chronische Verläufe zeigt. Neben den Lungen als primärem Manifestationsort können auch die Nieren, Knochen und andere Organe befallen sein. Eine von drei Personen weltweit ist mit Mycobacterium tuberculosis infiziert, insgesamt 2 Milliarden Menschen. In HIV/TB Co-Inifzierten Menschen entsteht ein komplexes Zusammenspiel zwischen HIV und M. tuberculosis, welches zu einer Verschlechterung beider Krankheitsbilder führt. HIV führt durch endogene Rekativierung oder exogene Re-Infektion zu einer Progression der Tuberkulose, welche im weiteren Verlauf die Krankheitsprogression von HIV beschleunigt. Sowohl Morbidität als auch Mortalität sind in HIV-1/TB Co-Infizierten Menschen erhöht. Aktive Lungentuberkulose und Miliartuberkulose gehen mit dem Anstieg der Diversifität der HIV Viren innerhalb eines Wirtes einher. Wie lange diese erhöhte Heterogenität der HIV Quasispecies nach der erfolgreichen Behandlung einer Tuberkulose bestehen bleibt ist bisher noch unklar. Das Verständnis des dem Zusammenspiel von HIV und TB zugrundeliegenden Mechanismus ist essentiell für die Entwicklung von effektiven Massnahmen zur Senkung der Morbidität und Mortalität in HIV/TB Co-infizierten Menschen. Die gegenwärtige Forschungsarbeit folgte daher der Frage, ob wärend einer aktiven TB Infektion eine Zunahme der Diversität der HIV-1 Quasispecies zu beobachten ist und ob diese Diversität während einer TB Therapie erhalten bleibt oder sich zurück bildet. Hierfür wurden die HIV-1 Quasispecies zu zwei Zeitpunkten untersucht, wobei Proben von HIV-1 infizierten Patienten mit aktiver Tuberkulose (HIV-1/TB) und HIV infizierte Patienten ohne Tuberkulose (HIV-1/non TB) verglichen wurden. Aus Plasmaproben der Frankfurter HIV Cohorte wurde HIV-1 RNA isoliert. C2V5 env wurde durch PCR amplifiziert und molekular cloniert. Acht bis fünfundzwanzig Clone wurden für jeden Patienten sequenziert. Mehrere phylogenetische Analysen wurden durchgeführt, welche tree inferences, Intra-Patienten- und virale Diversität und Divergenz, Selektionsdruckanalysen, Vorhersage der Co-Rezeptornutzung sowie Zweipunktanalysen der Identität von Quasispecies mit Hilfe des Mantel’s Test miteinschlossen. Die Analysen ergaben die folgenden Ergebnisse: 1) Eine aktive TB erhält die Diversität von HIV-1 Quasispecies über einen längeren Zeitraum. 2. Eine aktive TB verstärkt die HIV -1 Divergenz 3) TB könnte zu einer langsameren Evolution von X4 Varianten führen. Schlussfolgerung: eine aktive TB beeinflusst die Entwicklung der viralen Diversität und Divergenz von HIV-1 im Verlauf der Krankheit. Der Einfluss der aktiven TB auf die longitudinale Evolution von HIV-1 könnte insbesondere R5 Viren betreffen. Der Einsatz von CCR5-Corezeptor Inhibitoren in HIV-1/TB coinifizerten Patienten sollte daher in Langzeitstudien untersucht werden
Structural properties of food proteins underlying stability or susceptibility to human gastrointestinal digestion
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