6 research outputs found
Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils
Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B
Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils
Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B
Endurvirkjun á súlfatasafjölvirkni í kuldavirkum alkalískum fosfatasa úr Vibrio örveru
Modern enzymes have primarily one specific function. However, most enzymes contain remnants of catalytic activity of another type from their evolutionary past in a suppressed form. These evolutionary remnants can be divided into promiscuous activity and dormant activity, depending on whether these remnants still catalyse chemical reactions (to a measurable degree) or whether the necessary residues are merely present, but the enzymes require a minor adjustment to become active again. Promiscuous activity has long been considered important for protein evolution and this may also apply to dormant activity.
The hypothesis of this project is that it is possible to re-awaken the dormant activity of an enzyme and, thus, turn it into a promiscuous enzyme. The aim was to re-awaken arylsulfatase activity in the Vibrio alkaline phosphatase (VAP). Computational calculations were used to compare the electrostatic potential in E. coli alkaline phosphatase (has sulfatase activity) with the electrostatic potential of VAP. Our preliminary results confirmed that the sulfatase activity of wild-type VAP is negligible compared to that of E. coli alkaline phosphatase. Computational calculations were used to compare the electrostatic potential in E. coli alkaline phosphatase (has sulfatase activity) with the electrostatic potential of VAP. According to our calculations, four mutations (T112A/R113E/W274K/H116D) are necessary to revive promiscuous activity in VAP. In this project the mutants containing the W274K and T112A/R113E/W274K mutations were studied. The preliminary results showed clearly detectable promiscuous activity in the T112A/R113E/W274K mutant. These results are promising.
Another hypothesis was that the re-awakening of sulfatase activity would also re-awaken β-lactamase activity in VAP. The mutants that were studied did not show detectable β-lactamase activity. Furthermore, in addition to measuring their promiscuous activity, the kinetic behaviour and heat stability of the mutants and the wild-type enzyme were studied.
Finally, the effects of a number of different variables on the kinetic behaviour and global structural heat stability of VAP were studied. The results indicate that the length of the amino acid connection between the Strep-tag and the C-terminus, and the concentration of ethylene glycol and MgCl2, have significant effects on the kinetic behaviour of the enzyme. The results also suggest that the Tm value of VAP is significantly higher in a buffer containing low concentration (1 mM) of MgCl2 or MgSO4, than in a buffer without MgCl2 or MgSO4.Nútíma ensím hafa nær einungis eina sértæka virkni. Í flestum ensímum er hinsvegar að finna leifar af annarskonar virkni í bældu formi, sem er arfur frá frumensímum. Þessum hvötunarleifum er hægt að skipta upp í fjölvirkni (e. promiscuous activity) og sofandi virkni (e. dormant activity), eftir því hvort breiðari virkni er mælanleg í ensíminu eða hvort lítið þurfi að breytast í hvarfstöð ensímsins svo virknin birtist. Fjölvirkni hefur lengi verið talin mikilvæg fyrir þróun ensíma og það sama má segja um sofandi virkni.
Tilgáta þessa verkefnis var sú að hægt sé að breyta ensímum í fjölvirk ensím með því að vekja upp sofandi virkni sem er í dvala í ensímbyggingunni. Markmið þessa verkefnis var að vekja upp arylsúlfatasavirkni í alkalískum fosfatasa úr Vibrio örveru (VAP). Bráðabirgðaniðurstöður staðfestu að súlfatasavirkni VAP væri hverfandi samanborið við alkalískan fosfatasa úr E. coli. Samkvæmt tölvuútreikningum, þar sem rafsvið í alkalískum fosfatasa úr E. coli (sem er virkur súlfatasi) var borið saman við VAP, er nauðsynlegt að framkvæma fjórar stökkbreytingar (T112A/R113E/W274K/H116D) til þess endurvirkja súlfatasafjölvirkni í VAP. Í þessu verkefni voru aðeins skoðuð tvö stökkbrigði, W274K og T112A/R113E/W274K. Niðurstöðurnar sýndu fram á greinilega súlfatasafjölvirkni í T112A/R113E/W274K stökkbrigðinu. Þessar niðurstöður lofa góðu.
Önnur tilgáta er sú að með því að endurvekja súlfatasavirkni myndi einnig vakna upp β-lactamasavirkni í VAP. Stökkbrigðin sýndu enga sýnilega β-lactamasavirkni. Auk þess að skoða fjölþreifni stökkbrigðanna, var hvötunargeta og hitastöðugleiki þeirra rannsakaður.
Áhrif nokkurra mismunandi þátta á hvötunargetu og hitastöðugleika VAP voru einnig rannsökuð. Niðurstöðurnar sýndu fram á að lengdin á amínósýrutengingunni á milli Strep-tag og C-enda ensímsins, og styrkur ethylen glýkóls og MgCl2, hefur marktæk áhrif á hvötunargetu ensímsins. Niðurstöðurnar bentu einnig til þess að Tm gildi VAP væri hærra í buffer sem inniheldur lágan styrk (1 mM) af MgCl2 eða MgSO4 en í buffer sem inniheldur ekki MgCl2 eða MgSO4
Vímuefnaneysla í æð: Rannsókn á notuðum sprautunálum í Reykjavík
Vímuefnaneysla í æð er veigamikill áhrifaþáttur í ótímabærum veikindum á heimsvísu. Rannsóknir á vímuefnaneyslu í æð byggja að miklu leyti á frásögnum einstaklinga á meðferðarstofnunum, lítið er því vitað um þá sem nota vímuefni í æð en leita sér ekki lækninga. Rannsóknin byggir á evrópskri samvinnurannsókn (ESCAPE) sem sett var á laggirnar til að fylgjast með hvaða vímuefni finnast í notuðum sprautubúnaði með efnafræðilegri greiningu. Með aukinni vitneskju um hvaða vímuefni eru í notkun má bæta skaðaminnkandi þjónustu við einstaklinga sem nota vímuefni í æð og draga úr skaðlegum áhrifum neyslunnar. Meginmarkmið rannsóknarinnar var að skoða hvaða vímuefni finnast í notuðum sprautubúnaði sem skilað er til förgunar, hvaða efni er algengast að finna og algengi þess að fleiri en eitt efni finnist í hverri sprautu.
Efniviður og aðferðir: Notuðum sprautubúnaði var safnað frá október 2021 til mars 2022 í Reykjavík. Engin samskipti voru milli notenda sprautubúnaðarins og þess sem aflaði gagna. Ein sprauta var tekin úr hverju nálaboxi til að lágmarka líkur á að fleiri en eitt sýni fengist frá sama einstaklingi. Alls var 203 sýnum var safnað á tímabilinu. Eftir sýnameðhöndlun voru efnin greind með gasgreini sem tengdur er massaskynjara (GC/MS) og niðurstöður metnar með Masshunter hugbúnaði með aðstoð staðalefna helstu fíkniefna á markaði.
Niðurstöður: Alls fundust 10 mismunandi vímuefni og fjögur mismunandi íblöndunarefni í sprautunum. Metýlfenídat var algengasta vímuefnið en það fannst í 105 sprautum (51,7%). Önnur örvandi efni fundust í um 42% sprauta og ópíóíðar í um þriðjungi sprauta. Engin efni fundust í þremur sprautum. Algengasta íblöndunarefnið var koffín en það fannst í 86 sprautum (42,4%). Rúmlega helmingur sprauta (54,2%) innihélt tvö eða fleiri vímuefni eða íblöndunarefni.
Ályktanir: Í Reykjavík er metýlfenídat mun algengara til vímuefnaneyslu í æð samanborið við niðurstöður ESCAPE. Heróín, búprenorfín og katínónar voru með algengustu efnum í ESCAPE en fundust ekki í Reykjavík. Neyslan virðist blönduð, um helmingur sprauta í ESCAPE innihélt fleiri en eitt vímuefni en tæplega þriðjungur í Reykjavík. Algengt var að sjá blöndu með örvandi vímuefni og ópíóíða en einnig blöndu örvandi vímuefna. Íblöndunarefni eru algeng og ekki er víst að notendur séu meðvitaðir um það
Photo-Induced Cross-Linking of Unmodified α-Synuclein Oligomers
Photo-induced cross-linking of unmodified proteins (PICUP) has been used in the past to study size distributions of protein assemblies. PICUP may, for example, overcome the significant experimental challenges related to the transient nature, heterogeneity, and low concentration of amyloid protein oligomers relative to monomeric and fibrillar species. In the current study, a reaction chamber was designed, produced, and used for PICUP reaction optimization in terms of reaction conditions and lighting time from ms to s. These efforts make the method more reproducible and accessible and enable the use of shorter reaction times compared to previous studies. We applied the optimized method to an α-synuclein aggregation time course to monitor the relative concentration and size distribution of oligomers over time. The data are compared to the time evolution of the fibril mass concentration, as monitored by thioflavin T fluorescence. At all time points, the smaller the oligomer, the higher its concentration observed after PICUP. Moreover, the total oligomer concentration is highest at short aggregation times, and the decline over time follows the disappearance of monomers. We can therefore conclude that these oligomers form from monomers
Single-vesicle intensity and colocalization fluorescence microscopy to study lipid vesicle fusion, fission, and lipid exchange
Interactions of lipid vesicles play important roles in a large variety of functions and dysfunctions in the human body. Vital for several biochemical functions is the interaction between monomeric proteins and lipid membranes, and the induced phenomena such as fusion between vesicles and cell membranes, lipid exchange between the membranes, or vesicle fission. Identification of single events and their frequency of occurrence would provide valuable information about protein-lipid interactions in both healthy and degenerative pathways. In this work, we present a single-vesicle intensity and colocalization fluorescence microscopy assay with a custom-written MATLAB analysis program. The assay can be used to study lipid exchange as well as vesicle fusion and fission between two vesicle populations labeled with different fluorescent dyes. Vesicles from the two populations are first mixed and docked to a glass surface. The sample is then simultaneously imaged using two separate wavelength channels monitoring intensity changes and colocalization of vesicles from the two populations. The monomeric pre-synaptic protein α-synuclein (α-syn) and small unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DOPS), and monosialotetrahexosylganglioside (GM1) were used as a model system to evaluate the method. From our analysis, neither α-syn induced fusion nor lipid exchange was observed for vesicles consisting of DOPC:DOPS (7:3). However, including 10% GM1 in the vesicles resulted in a 91% increase of the number of vesicles within 10 min, combined with a 57% decrease in the average fluorescence intensity per vesicle, indicating that approximately half of the vesicles underwent fission. The method facilitates the study of lipid vesicle fusion, fission, and lipid exchange under controlled conditions. It also allows these events to be studied for systems with more complex composition including exosomes and lipid-based drug carriers, to enable a better understanding of their physicochemical properties