How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

2008 ◽  
Vol 415 (2) ◽  
pp. 275-288 ◽  
Author(s):  
Satish Kumar ◽  
Vijay Kumar Ravi ◽  
Rajaram Swaminathan
Keyword(s):  
Disulfide Bonds ◽  
Molecular Mechanisms ◽  
Amyloid Fibrils ◽  
Gel Filtration ◽  
Segmental Motion ◽  
Initial Growth ◽  
Time Resolved ◽  
Force Microscopy ◽  
Intermolecular Disulfide Bonds ◽  
Soluble Aggregates

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0–24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (∼5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1–2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.

scholarly journals Stabilization, Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

2013 ◽  
Vol 288 (23) ◽  
pp. 16438-16450 ◽  
Author(s):  
Gustav Östner ◽  
Veronica Lindström ◽  
Per Hjort Christensen ◽  
Maciej Kozak ◽  
Magnus Abrahamson ◽  
...  
Keyword(s):  
Cystatin C ◽  
Amyloid Fibrils ◽  
Dynamic Equilibrium ◽  
Biological Fluids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Domain Swapping ◽  
Amyloid Angiopathy ◽  
Force Microscopy ◽  
And Function

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

scholarly journals In Situ Initial Growth Studies of SrTiO3 on SrTiO3 by Time Resolved High Pressure RHEED

1998 ◽  
Vol 526 ◽  
Author(s):  
Gertjan Koster ◽  
Guus J.H.M. Rijnders ◽  
Dave H.A. Blank ◽  
Horst Rogalla
Keyword(s):  
High Pressure ◽  
Pulsed Laser ◽  
High Energy ◽  
Initial Growth ◽  
Time Resolved ◽  
Force Microscopy ◽  
Atomic Force ◽  
Different Temperatures ◽  
Substrate Surfaces

AbstractThe initial growth of pulsed laser deposited SrTiO3 on SrTiO3 has been studied using high pressure Reflection High Energy Electron Diffraction (RHEED) and Atomic Force Microscopy (AFM). For this, we developed a Pulsed Laser Deposition (PLD)-RHEED system, with the possibility to study the growth and to monitor the growth rates, in situ, at typical PLD pressures (10-50 Pa). Using perfect single crystal SrTiO3 substrate surfaces, we observe true 2D intensity oscillations at different temperatures. Simultaneously, information on the diffusion of the deposited material on the surface could be extracted from the relaxation of the intensity after each laser pulse. The characteristic times depend on pressure and temperature as well as the 2D coverage during growth.

A combined approach to characterize ligand-induced solid–solid phase transitions in biomacromolecular crystals

2021 ◽  
Vol 54 (3) ◽  
Author(s):  
Saminathan Ramakrishnan ◽  
Jason R. Stagno ◽  
Valentin Magidson ◽  
William F. Heinz ◽  
Yun-Xing Wang
Keyword(s):  
Phase Transitions ◽  
Conformational Changes ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Solid Phase ◽  
Control Element ◽  
Time Resolved ◽  
Crystalline Materials ◽  
Force Microscopy ◽  
Glass Surfaces

Solid–solid phase transitions (SSPTs) are widespread naturally occurring phenomena. Understanding the molecular mechanisms and kinetics of SSPTs in various crystalline materials, however, has been challenging due to technical limitations. In particular, SSPTs in biomacromolecular crystals, which may involve large-scale changes and particularly complex sets of interactions, are largely unexplored, yet may have important implications for time-resolved crystallography and for developing synthetic biomaterials. The adenine riboswitch (riboA) is an RNA control element that uses ligand-induced conformational changes to regulate gene expression. Crystals of riboA, upon the addition of a ligand, undergo an SSPT from monoclinic to triclinic to orthorhombic. Here, solution atomic force microscopy (AFM) and polarized video microscopy (PVM) are used to characterize the multiple transition states throughout the SSPT in both the forward and the reverse directions. This contribution describes detailed protocols for growing crystals directly on mica or glass surfaces for AFM and PVM characterization, respectively, as well as methods for image processing and phase-transition kinetics analysis.

scholarly journals Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

2015 ◽  
Vol 112 (16) ◽  
pp. 4946-4951 ◽  
Author(s):  
Katy N. Olafson ◽  
Megan A. Ketchum ◽  
Jeffrey D. Rimer ◽  
Peter G. Vekilov
Keyword(s):  
Antimalarial Drug ◽  
Molecular Mechanisms ◽  
Physiological Concentration ◽  
Time Resolved ◽  
Force Microscopy ◽  
Atomic Structures ◽  
Future Design ◽  
Surface Sites ◽  
Atomic Force

Hematin crystallization is the primary mechanism of heme detoxification in malaria parasites and the target of the quinoline class of antimalarials. Despite numerous studies of malaria pathophysiology, fundamental questions regarding hematin growth and inhibition remain. Among them are the identity of the crystallization medium in vivo, aqueous or organic; the mechanism of crystallization, classical or nonclassical; and whether quinoline antimalarials inhibit crystallization by sequestering hematin in the solution, or by blocking surface sites crucial for growth. Here we use time-resolved in situ atomic force microscopy (AFM) and show that the lipid subphase in the parasite may be a preferred growth medium. We provide, to our knowledge, the first evidence of the molecular mechanisms of hematin crystallization and inhibition by chloroquine, a common quinoline antimalarial drug. AFM observations demonstrate that crystallization strictly follows a classical mechanism wherein new crystal layers are generated by 2D nucleation and grow by the attachment of solute molecules. We identify four classes of surface sites available for binding of potential drugs and propose respective mechanisms of drug action. Further studies reveal that chloroquine inhibits hematin crystallization by binding to molecularly flat {100} surfaces. A 2-μM concentration of chloroquine fully arrests layer generation and step advancement, which is ∼104× less than hematin’s physiological concentration. Our results suggest that adsorption at specific growth sites may be a general mode of hemozoin growth inhibition for the quinoline antimalarials. Because the atomic structures of the identified sites are known, this insight could advance the future design and/or optimization of new antimalarials.

scholarly journals Review of time-resolved non-contact electrostatic force microscopy techniques with applications to ionic transport measurements

2019 ◽  
Vol 10 ◽  
pp. 617-633 ◽  
Author(s):  
Aaron Mascaro ◽  
Yoichi Miyahara ◽  
Tyler Enright ◽  
Omur E Dagdeviren ◽  
Peter Grütter
Keyword(s):  
Atomic Force Microscopy ◽  
Electrostatic Force ◽  
Oscillation Period ◽  
Electrostatic Force Microscopy ◽  
Time Varying ◽  
Time Resolved ◽  
Battery Materials ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Techniques

Recently, there have been a number of variations of electrostatic force microscopy (EFM) that allow for the measurement of time-varying forces arising from phenomena such as ion transport in battery materials or charge separation in photovoltaic systems. These forces reveal information about dynamic processes happening over nanometer length scales due to the nanometer-sized probe tips used in atomic force microscopy. Here, we review in detail several time-resolved EFM techniques based on non-contact atomic force microscopy, elaborating on their specific limitations and challenges. We also introduce a new experimental technique that can resolve time-varying signals well below the oscillation period of the cantilever and compare and contrast it with those previously established.

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