scholarly journals Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of β-Annulus Peptide into Artificial Viral Capsid

2021 ◽  
Vol 22 (9) ◽  
pp. 4754
Author(s):  
Risako Kobayashi ◽  
Hiroshi Inaba ◽  
Kazunori Matsuura
Keyword(s):  
Self Assembly ◽  
Fluorescence Correlation Spectroscopy ◽  
De Novo ◽  
Correlation Spectroscopy ◽  
Viral Capsid ◽  
Molecular Crowding ◽  
Viral Capsids ◽  
Fluorescence Correlation ◽  
Self Assembling ◽  
Dilute Aqueous Solutions

Recent progress in the de novo design of self-assembling peptides has enabled the construction of peptide-based viral capsids. Previously, we demonstrated that 24-mer β-annulus peptides from tomato bushy stunt virus spontaneously self-assemble into an artificial viral capsid. Here we propose to use the artificial viral capsid through the self-assembly of β-annulus peptide as a simple model to analyze the effect of molecular crowding environment on the formation process of viral capsid. Artificial viral capsids formed by co-assembly of fluorescent-labelled and unmodified β-annulus peptides in dilute aqueous solutions and under molecular crowding conditions were analyzed using fluorescence correlation spectroscopy (FCS). The apparent particle size and the dissociation constant (Kd) of the assemblies decreased with increasing concentration of the molecular crowding agent, i.e., polyethylene glycol (PEG). This is the first successful in situ analysis of self-assembling process of artificial viral capsid under molecular crowding conditions.

Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding

Soft Matter ◽  
2009 ◽  
Vol 5 (7) ◽  
pp. 1358 ◽  
Author(s):  
Claus B. Müller ◽  
Thomas Eckert ◽  
Anastasia Loman ◽  
Jörg Enderlein ◽  
Walter Richtering
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Molecular Crowding ◽  
Fluorescence Correlation

scholarly journals Deciphering Molecular Self-Assembly through Electron Microscopy and Fluorescence Correlation Spectroscopy

2020 ◽  
Author(s):  
Subhankar Kundu ◽  
Arkaprava Chowdhury, ◽  
Somen Nandi ◽  
Kankan Bhattacharyya ◽  
Abhijit Patra
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
The Self ◽  
Fine Tuning ◽  
Assembly Process ◽  
Molecular Aggregates ◽  
Fluorescence Correlation ◽  
Self Assembled

Supramolecular self-assembly of small organic molecules has emerged as a powerful tool to construct well-defined micro- and nanoarchitecture through fine-tuning a range of intermolecular interactions. The size, shape, and optical properties of these nanostructures largely depend on the temperature and polarity of the medium, along with the specific self-assembled pattern of molecular building units. The engineering of supramolecular self-assembled nanostructures with morphology-dependent tunable emission is in high demand due to the promising scope in nanodevices and molecular machines. However, challenges are probing the evolution of molecular aggregates from a true solution and directing the self-assembly process in a pre-defined fashion. The structure of molecular aggregates in the solution can be predicted from fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) analysis. On the other hand, the morphology of the aggregates can also be visualized through electron microscopy. Nevertheless, a direct correlation between emission from molecular aggregates in the aqueous dispersion and their morphology obtained through a solid-state characterization is missing. In the present study, we decipher the sequential evolution of molecular nanofibers from solution to spherical and oblong-shaped nanoparticles through the variation of solvent polarity, adjusting the <a>hydrophobic-hydrophilic interactions</a>. The intriguing case of molecular self-assembly is elucidated employing a newly designed π-conjugated thiophene derivative (TPAn) through a combination of steady-state absorption, emission measurements, FCS, and electron microscopy. The FCS analysis and microscopy results infer that small-sized nanofibers in the dispersion are further agglomerated, resulting in a network of nanofibers upon solvent evaporation. <a>The evolution of organic nanofibers and subtle control over the self-assembly process demonstrated in the current investigation provides a general paradigm to correlate the size, shape, and emission properties of diverse fluorescent molecular aggregates in complex heterogeneous media, including a human cell. </a>

scholarly journals Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

2015 ◽  
Vol 2 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Cornelia Junghans ◽  
Franz-Josef Schmitt ◽  
Vladana Vukojević ◽  
Thomas Friedrich
Keyword(s):  
Membrane Proteins ◽  
Fluorescence Correlation Spectroscopy ◽  
Chemical Properties ◽  
Cholesterol Content ◽  
Correlation Spectroscopy ◽  
Molecular Crowding ◽  
Diffusion Behavior ◽  
Temporal Precision ◽  
Fluorescence Correlation ◽  
Quenching Process

AbstractMeasurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1– 100 ms). Since the origin of this quenching process is currently unclear, care has to be taken when the Dreiklang label is intended to be used in FCS applications.

scholarly journals Deciphering Molecular Self-Assembly through Electron Microscopy and Fluorescence Correlation Spectroscopy

2020 ◽  
Author(s):  
Subhankar Kundu ◽  
Arkaprava Chowdhury, ◽  
Somen Nandi ◽  
Kankan Bhattacharyya ◽  
Abhijit Patra
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
The Self ◽  
Fine Tuning ◽  
Assembly Process ◽  
Molecular Aggregates ◽  
Fluorescence Correlation ◽  
Self Assembled

Supramolecular self-assembly of small organic molecules has emerged as a powerful tool to construct well-defined micro- and nanoarchitecture through fine-tuning a range of intermolecular interactions. The size, shape, and optical properties of these nanostructures largely depend on the temperature and polarity of the medium, along with the specific self-assembled pattern of molecular building units. The engineering of supramolecular self-assembled nanostructures with morphology-dependent tunable emission is in high demand due to the promising scope in nanodevices and molecular machines. However, challenges are probing the evolution of molecular aggregates from a true solution and directing the self-assembly process in a pre-defined fashion. The structure of molecular aggregates in the solution can be predicted from fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) analysis. On the other hand, the morphology of the aggregates can also be visualized through electron microscopy. Nevertheless, a direct correlation between emission from molecular aggregates in the aqueous dispersion and their morphology obtained through a solid-state characterization is missing. In the present study, we decipher the sequential evolution of molecular nanofibers from solution to spherical and oblong-shaped nanoparticles through the variation of solvent polarity, adjusting the <a>hydrophobic-hydrophilic interactions</a>. The intriguing case of molecular self-assembly is elucidated employing a newly designed π-conjugated thiophene derivative (TPAn) through a combination of steady-state absorption, emission measurements, FCS, and electron microscopy. The FCS analysis and microscopy results infer that small-sized nanofibers in the dispersion are further agglomerated, resulting in a network of nanofibers upon solvent evaporation. <a>The evolution of organic nanofibers and subtle control over the self-assembly process demonstrated in the current investigation provides a general paradigm to correlate the size, shape, and emission properties of diverse fluorescent molecular aggregates in complex heterogeneous media, including a human cell. </a>

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