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

ABSTRACT: 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.

SUBMITTER: Ramakrishnan S

PROVIDER: S-EPMC8202036 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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

Ramakrishnan Saminathan S Stagno Jason R JR Magidson Valentin V Heinz William F WF Wang Yun-Xing YX

Journal of applied crystallography 20210509 Pt 3

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 b ...[more]

PMID: 34194289

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Project description:We report that phospholipid vesicles incorporating ligands, when captured from solution onto surfaces presenting receptors for these ligands, can trigger surface-induced orientational ordering transitions in nematic phases of 4'-pentyl-4-cyanobiphenyl (5CB). Specifically, whereas avidin-functionalized surfaces incubated against vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were observed to cause the liquid crystal (LC) to adopt a parallel orientation at the surface, the same surfaces incubated against biotinylated vesicles (DOPC and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DOPE)) caused the homeotropic (perpendicular) ordering of the LC. The use of a combination of atomic force microscopy (AFM), ellipsometry and quantitative fluorimetry, performed as a function of vesicle composition and vesicle concentration in solution, revealed the capture of intact vesicles containing 1% biotin-DOPE from buffer at the avidin-functionalized surfaces. Subsequent exposure to water prior to contact with the LC, however, resulted in the rupture of the majority of vesicles into interfacial multilayer assemblies with a maximum phospholipid loading set by random close packing of the intact vesicles initially captured on the surface (5.1 ± 0.2 phospholipid molecules/nm(2)). At high concentrations of biotinylated lipid (>10% biotin-DOPE) in the vesicles, the limiting lipid loading was measured to be 4.0 ± 0.3 phospholipid molecules/nm(2), consistent with the maximum phospholipid loading set by the spontaneous formation of a bilayer during incubation with the biotinylated vesicles. We measured the homeotropic ordering of the LC on the surfaces independently of the initial morphology of the phospholipid assembly captured on the surface (intact vesicle, planar multilayer). We interpret this result to infer the reorganization of the phospholipid bilayers either prior to or upon contact with the LCs such that interactions of the acyl chains of the phospholipid and the LC dominate the ordering of the LC, a conclusion that is further supported by quantitative measurements of the orientation of the LC as a function of the phospholipid surface density (>1.8 molecules/nm(2) is required to cause the homeotropic ordering of the LC). These results and others presented herein provide fundamental insights into the interactions of phospholipid-decorated interfaces with LCs and thereby provide guidance for the design of surfaces on which phospholipid assemblies captured through ligand-receptor recognition can be reported via ordering transitions in LCs.

Dataset Information

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

Publications

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

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