| 1. |
- Clifton, Luke A., et al.
(author)
-
Creation of distinctive Bax-lipid complexes at mitochondrial membrane surfaces drives pore formation to initiate apoptosis
- 2023
-
In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:22
-
Journal article (peer-reviewed)abstract
- Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of whichwere critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death.
|
|
| 2. |
- Clifton, Luke A, et al.
(author)
-
Design and use of model membranes to study biomolecular interactions using complementary surface-sensitive techniques.
- 2020
-
In: Advances in Colloid and Interface Science. - : Elsevier. - 0001-8686 .- 1873-3727. ; 277
-
Journal article (peer-reviewed)abstract
- Cellular membranes are complex structures and simplified analogues in the form of model membranes or biomembranes are used as platforms to understand fundamental properties of the membrane itself as well as interactions with various biomolecules such as drugs, peptides and proteins. Model membranes at the air-liquid and solid-liquid interfaces can be studied using a range of complementary surface-sensitive techniques to give a detailed picture of both the structure and physicochemical properties of the membrane and its resulting interactions. In this review, we will present the main planar model membranes used in the field to date with a focus on monolayers at the air-liquid interface, supported lipid bilayers at the solid-liquid interface and advanced membrane models such as tethered and floating membranes. We will then briefly present the principles as well as the main type of information on molecular interactions at model membranes accessible using a Langmuir trough, quartz crystal microbalance with dissipation monitoring, ellipsometry, atomic force microscopy, Brewster angle microscopy, Infrared spectroscopy, and neutron and X-ray reflectometry. A consistent example for following biomolecular interactions at model membranes is used across many of the techniques in terms of the well-studied antimicrobial peptide Melittin. The overall objective is to establish an understanding of the information accessible from each technique, their respective advantages and limitations, and their complementarity.
|
|
| 3. |
- Clifton, Luke A., et al.
(author)
-
Insight into Bcl-2 proteins' functioning at mitochondrial membrane level
- 2023
-
In: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 122:3S1, s. 232a-232a
-
Journal article (peer-reviewed)abstract
- Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes.
|
|
| 4. |
- Correa, Yubexi, et al.
(author)
-
Lipid exchange of apolipoprotein A-I amyloidogenic variants in reconstituted high-density lipoprotein with artificial membranes
- 2024
-
In: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 33:5
-
Journal article (peer-reviewed)abstract
- High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.
|
|
| 5. |
- Correa, Yubexi, et al.
(author)
-
Lipid exchange of apolipoprotein A-I amyloidogenic variants in reconstituted high-density lipoprotein with artificial membranes
- 2024
-
In: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 33:5
-
Journal article (peer-reviewed)abstract
- High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid–protein interactions and their behavior.
|
|
| 6. |
|
|
| 7. |
- Duong, Vicky, et al.
(author)
-
Exploring translational gaps between basic scientists, clinical researchers, clinicians, and consumers : Proceedings and recommendations arising from the 2020 mine the gap online workshop
- 2021
-
In: Osteoarthritis and Cartilage Open. - : Elsevier BV. - 2665-9131. ; 3:2
-
Journal article (peer-reviewed)abstract
- Objective: To provide a summary of the translational gaps in musculoskeletal research as identified in the Mine the Gap workshop and propose possible solutions. Methods: The Mine the Gap online workshop was hosted on October 14th and 15th, 2020. Five international panels, each comprised of a clinician, clinical researcher and basic scientist, presented gaps and proposed solutions for the themes of biomechanics, pain, biological measurements, phenotypes and imaging. This was followed by an interactive panel discussion with consumer insights. Results: A number of translational gaps and proposed solutions across each of the five themes were identified. A consumer panel provided constructive feedback highlighting the need for improved resources, communication and shared decision making, and treatment individualisation. Conclusion: This brief report provides a greater understanding of the diverse work and gaps relevant to fundamental/discovery scientists, clinical researchers and clinicians working across the musculoskeletal field. The numerous translational gaps highlight the need to improve communication and collaboration across the musculoskeletal field.
|
|
| 8. |
- Hughes, Arwel, et al.
(author)
-
Physical Properties of Bacterial Outer Membrane Models : Neutron Reflectometry & Molecular Simulation
- 2019
-
In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 116:6, s. 1095-1104
-
Journal article (peer-reviewed)abstract
- The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM.
|
|
| 9. |
- Jo, Sunhwan, et al.
(author)
-
Multiple Conformational States Contribute to the 3D Structure of a Glucan Decasaccharide : A Combined SAXS and MD Simulation Study
- 2018
-
In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 122:3, s. 1169-1175
-
Journal article (peer-reviewed)abstract
- The inherent flexibility of carbohydrates is dependent on stereochemical arrangements, and characterization of their influence and importance will give insight into the three-dimensional structure and dynamics. In this study, a beta-(1 -> 4)/beta-(1 -> 3)-linked glucosyl decasaccharide is experimentally investigated by synchrotron small-angle X-ray scattering from which its radius of gyration (R-g) is obtained. Molecular dynamics (MD) simulations of the decasaccharide show four populated states at each glycosidic linkage, namely, syn- and anti-conformations. The calculated R-g values from the MD simulation reveal that in addition to syn-conformers the presence of anti-psi conformational states is required to reproduce experimental scattering data, unveiling inherent glycosidic linkage flexibility. The CHARMM36 force field for carbohydrates thus describes the conformational flexibility of the decasaccharide very well and captures the conceptual importance that anti-conformers are to be anticipated at glycosidic linkages of carbohydrates.
|
|
| 10. |
- Lakey, Jeremy H., et al.
(author)
-
Exploiting neutron scattering contrast variation in biological membrane studies
- 2022
-
In: Biophysics Reviews. - : American Institute of Physics (AIP). - 2688-4089. ; 3:2
-
Research review (peer-reviewed)abstract
- Biological membranes composed of lipids and proteins are central for the function of all cells and individual components, such as proteins, that are readily studied by a range of structural approaches, including x-ray crystallography and cryo-electron microscopy. However, the study of complex molecular mixtures within the biological membrane structure and dynamics requires techniques that can study nanometer thick molecular bilayers in an aqueous environment at ambient temperature and pressure. Neutron methods, including scattering and spectroscopic approaches, are useful since they can measure structure and dynamics while also being able to penetrate sample holders and cuvettes. The structural approaches, such as small angle neutron scattering and neutron reflectometry, detect scattering caused by the difference in neutron contrast (scattering length) between different molecular components such as lipids or proteins. Usually, the bigger the contrast, the clearer the structural data, and this review uses examples from our research to illustrate how contrast can be increased to allow the structures of individual membrane components to be resolved. Most often this relies upon the use of deuterium in place of hydrogen, but we also discuss the use of magnetic contrast and other elements with useful scattering length values.
|
|
