| 1. |
- Hariharan, Parameswaran, et al.
(författare)
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Thermodynamics of Nanobody Binding to Lactose Permease
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:42, s. 5917-5926
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Tidskriftsartikel (refereegranskat)abstract
- Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure.
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| 2. |
- Kelly, John J., et al.
(författare)
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Snapshots of actin and tubulin folding inside the TRiC chaperonin
- 2022
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Ingår i: Nature Structural and Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 29:5, s. 420-429
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Tidskriftsartikel (refereegranskat)abstract
- The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.
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| 3. |
- Löw, Christian, et al.
(författare)
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Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:10, s. e77984-
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Tidskriftsartikel (refereegranskat)abstract
- Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.
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| 4. |
- Miremadi, S. Reza, et al.
(författare)
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A randomized controlled trial comparing surgical and non-surgical periodontal therapy : a 3-year clinical and cost-effectiveness analysis
- 2015
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Ingår i: Journal of Clinical Periodontology. - : John Wiley & Sons. - 0303-6979 .- 1600-051X. ; 42:8, s. 748-755
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Tidskriftsartikel (refereegranskat)abstract
- AimTo compare immediate surgery to scaling and root planing (SRP) in the treatment of advanced periodontitis focusing on the prevalence of residual pockets after treatment and cost-effectiveness (1); to elucidate the adjunctive effects of azithromycin in a second-treatment phase (2) up to 36months. Materials and MethodsThirty-nine patients (18 males, 21 females; mean age: 54.6) received oral hygiene instructions and were randomly allocated to surgery (n=19) or SRP (n=20). Patients with residual pockets (6mm) at 6months received re-debridement of these sites and systemic azithromycin. Clinical measurements were performed at 12, 24 and 36months whereby residual pockets underwent re-debridement. Patients were evaluated in terms of clinical response parameters and cost-effectiveness. Chair-time was used to assess the financial impact of treatment. ResultsOnly six patients in the surgery group required additional treatment at 6months, whereas 14 patients in the SRP underwent this therapy. Both treatment arms were equally effective in terms of clinical outcome demonstrating <2% residual pockets at 36months. Surgery imposed an extra 746 Euro on the patient up to 6months compared to SRP. At 36months, 69 Euro of this amount could be offset due to the lower need for re-treatment during maintenance phase. ConclusionsSurgery was ultimately associated with a significantly lower need for re-treatment during supportive care, however, at significantly higher costs over the 3years.
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| 5. |
- Miremadi, Seyed Reza, et al.
(författare)
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A randomized controlled trial on immediate surgery versus root planing in patients with advanced periodontal disease : a cost-effectiveness analysis
- 2014
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Ingår i: Journal of Clinical Periodontology. - : John Wiley & Sons. - 0303-6979 .- 1600-051X. ; 41:2, s. 164-171
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Tidskriftsartikel (refereegranskat)abstract
- AimTo compare immediate surgery to scaling and root planing (SRP) in the treatment of advanced periodontal disease focusing on the prevalence of residual sites and cost-effectiveness (1); to evaluate the adjunctive effects of azithromycin in a second treatment phase (2). Materials and MethodsThirty-nine patients (18 males, 21 females; mean age: 54.6) received oral hygiene instructions and were randomly allocated to surgery (n=19) or SRP (n=20). Patients with residual pockets (6mm) at 6months received re-debridement of these sites and systemic azithromycin. Treatment groups were followed up to 12months and evaluated in terms of clinical response parameters and cost-effectiveness. Chair-time was used to assess the financial impact of treatment. ResultsBoth treatment arms were equally effective in terms of clinical outcome demonstrating less than 1% residual pockets at 12months. Surgery imposed an extra 746 Euro on the patient up to 6months when compared to SRP. At 12months, 46 Euro of this amount could be offset as a result of a reduced need for supportive care. Only 6 patients in the surgery group needed systemic antibiotics, whereas 14 patients in the SRP needed such additional treatment. ConclusionsAlthough 700 Euro could be saved on average by performing SRP instead of surgery, the latter significantly reduced the need for supportive care and systemic antibiotics.
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