41.
Blom, Anna, et al.
(författare)
Structural requirements for the complement regulatory activities of C4BP
2001
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:29, s. 27136-27144
Tidskriftsartikel (refereegranskat) abstract
C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity.
42.
Braun, Katharina, et al.
(författare)
Membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides
2016
Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 0021-9797 .- 1095-7103. ; 475, s. 161-170
Tidskriftsartikel (refereegranskat) abstract
Membrane interactions are critical for the successful use of mesoporous silica nanoparticles as delivery systems for antimicrobial peptides (AMPs). In order to elucidate these, we here investigate effects of nanoparticle charge and porosity on AMP loading and release, as well as consequences of this for membrane interactions and antimicrobial effects. Anionic mesoporous silica particles were found to incorporate considerable amounts of the cationic AMP LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37), whereas loading is much lower for non-porous or positively charged silica nanoparticles. Due to preferential pore localization, anionic mesoporous particles, but not the other particles, protect LL-37 from degradation by infection-related proteases. For anionic mesoporous nanoparticles, membrane disruption is mediated almost exclusively by peptide release. In contrast, non-porous silica particles build up a resilient LL-37 surface coating due to their higher negative surface charge, and display largely particle-mediated membrane interactions and antimicrobial effects. For positively charged mesoporous silica nanoparticles, LL-37 incorporation promotes the membrane binding and disruption displayed by the particles in the absence of peptide, but also causes toxicity against human erythrocytes. Thus, the use of mesoporous silica nanoparticles as AMP delivery systems requires consideration of membrane interactions and selectivity of both free peptide and the peptide-loaded nanoparticles, the latter critically dependent on nanoparticle properties.
43.
Carlsson, Lena, et al.
(författare)
Desmin-related myopathies in mice and man
2001
Ingår i: Acta Physiologica Scandinavica. - : Wiley-Blackwell. - 0001-6772 .- 1365-201X. ; 171:3, s. 341-8
Tidskriftsartikel (refereegranskat) abstract
Desmin, the main intermediate filament (IF) protein in skeletal and heart muscle cells, is of great importance as a part of the cytoskeleton. The IFs surround and interlink myofibrils, and connect the peripheral myofibrils with the sarcolemma. In myotendinous junctions and neuromuscular junctions of skeletal muscle fibres, desmin is enriched. In the heart, desmin is increased at intercalated discs, the attachment between cardiomyocytes, and it is the main component in Purkinje fibres of the conduction system. Desmin is the first muscle-specific protein to appear during myogenesis. Nevertheless, lack of desmin, as shown from experiments with desmin knockout (K/O) mice, does not influence myogenesis or myofibrillogenesis. However, the desmin knock-out mice postnatally develop a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. In other skeletal muscles the organization of myofibrils is remarkably unaffected. Thus, the main consequence of the lack of desmin is that the muscle fibres become more susceptible to damage. The loss of membrane integrity leads to a dystrophic process, with degeneration and fibrosis. In the heart cardiac failure develops, whereas in affected skeletal muscles regenerative attempts are seen. In humans, accumulations of desmin have been a hallmark for presumptive desmin myopathies. Recent investigations have shown that some families with such a myopathy have a defect in the gene coding for alphaB-crystallin, whereas others have mutations in the desmin gene. Typical features of these patients are cardiac affections and muscle weakness. Thus, mutations in the desmin gene is pathogenic for a distinct type of muscle disorder.
44.
Chapman, Henry N, et al.
(författare)
Femtosecond X-ray protein nanocrystallography.
2011
Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 470:7332, s. 73-7
Tidskriftsartikel (refereegranskat) abstract
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200nm to 2μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
45.
Dahlin, Andreas, 1980
(författare)
Nanoplasmonic Biosensors compatible with Artificial Cell Membranes
2008
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Within life science, there is currently an intense search for novel techniques that enable efficient and reliable analysis of biomolecular interactions. Such methods have future applications within medical diagnostics and drug development, as well as within proteomic research in general. Lately, several concepts have emerged that are based on monitoring molecular binding to surfaces via optical, mechanical or electrical signal transduction. In particular, the plasmons associated with metallic nanoparticles are of interest since they offer a convenient way to monitor biomolecular interactions, also in a miniaturized format, by optical spectroscopy.This thesis describes the development of a biosensor based on the optical properties of nanoscale apertures in continuous metal films. The fabrication and characterization of the nanostructure is described, as well as surface modification protocols based on thiol chemistry for material-specific functionalization. In addition, an experimental setup for spectroscopy is presented together with data analysis algorithms for minimizing noise.It is emphasized that, from an experimental sensing perspective, nanoholes and nanoparticles have essentially the same plasmonic properties. However, the nanoholes offer several advantages because of the fact that the structure is physically different. In particular, it is shown how various artificial cell membranes can be spontaneously formed inside nanoholes. This makes the sensor compatible with studies of processes related to biological membranes. In this context, membrane-bound proteins are of special interest since they constitute a third of our genome and represent the target of half of the most common medical drugs. Potential future applications of the artificial membranes on the plasmonic nanostructures are discussed, with focus on probing transport across the membrane.
46.
Danelius, Emma, et al.
(författare)
Flexibility is important for inhibition of the MDM2/p53 protein-protein interaction by cyclic β-hairpins
2016
Ingår i: Organic and Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 14, s. 10386-10393
Tidskriftsartikel (refereegranskat) abstract
© 2016 The Royal Society of Chemistry.Protein-protein interactions that have large, flat and featureless binding sites are difficult drug targets. In the development of their modulators conventional drug discovery strategies are often unsuccessful. Gaining a detailed understanding of the binding mode of protein-protein interaction inhibitors is therefore of vast importance for their future pharmaceutical use. The MDM2/p53 protein pair is a highly promising target for cancer treatment. Disruption of the protein complex using p53 α-helix mimetics has been shown to be a successful strategy to control p53 activity. To gain further insight into the binding of inhibitors to MDM2, the flexibility of four cyclic β-hairpins that act as α-helical mimetics and potential MDM2/p53 interaction inhibitors was investigated in relation to their inhibitory activity. MDM2-binding of the mimetics was determined using fluorescence polarization and surface plasmon resonance assays, whereas their conformation and dynamics in solution was described by the combined experimental and computational NAMFIS analysis. Molecular flexibility was shown to be important for the activity of the cyclic β-hairpin based MDM2 inhibitors.
47.
48.
Engell, Maria Terese, et al.
(författare)
Postural changes and their effects in elite riders when actively influencing the horse versus sitting passively at trot
2016
Ingår i: Comparative Exercise Physiology. - 1755-2540 .- 1755-2559. ; 12, s. 27-33
Tidskriftsartikel (refereegranskat) abstract
The objectives were to compare sagittal plane posture of the pelvis, trunk and head of elite dressage riders when they ride actively to train the horse versus sitting passively and following the horses' movements at trot, and to evaluate the effects of these changes in rider posture on load distribution on the horse's back. Synchronised motion capture and saddle mat data of seven elite dressage riders were used to measure minimal and maximal angles and range of motion (ROM) for the pelvic, trunk and head segments, the angle between pelvis and trunk segments, phase-shift between pitching motions of pelvis and trunk, and pelvic translation relative to the saddle. Non-parametric statistical tests compared variables between the two rider postures. In the passive rider posture the pelvis, trunk and head showed two pitching cycles per stride. Maximal posterior and anterior pelvic rotation occurred, respectively, early and late in the horse's diagonal stance phase. Compared with pelvic movements, trunk movements were slightly delayed and head movements were out-of-phase. In the active rider posture the pelvis and trunk pitched further posteriorly throughout the stride. Most of the riders showed similar sagittal plane movements of the axial body segments but with some notable individual variations.
49.
Fleming, Cassandra L., et al.
(författare)
On-Command Regulation of Kinase Activity using Photonic Stimuli
2019
Ingår i: ChemPhotoChem. - : Wiley. - 2367-0932. ; 3:6, s. 318-326
Forskningsöversikt (refereegranskat) abstract
The underlying role that many kinases play in complex cellular pathways as well as disease remains unclear. To better understand the role that kinases play in both health and disease states, the use of light as an external stimulus to modulate kinase activity with high spatiotemporal resolution has gained increasing interest over the years. Herein we highlight the progress made towards the development of light-responsive kinase enzymes and small molecule inhibitors. In these examples, photolabile caging groups and photoswitchable entities have been utilised to modulate either kinase activation or inhibition in a light-controlled manner.
50.
Friedrich, Ute, et al.
(författare)
Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C
2001
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:26, s. 24122-24128
Tidskriftsartikel (refereegranskat) abstract
Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC.
