NMR Detection of an Equilibrium Phase Consisting of Monomers and Clusters in Concentrated Lysozyme Solutions

Barhoum, Suliman; Yethiraj, Anand

doi:10.1021/jp108995k

Cited by 27 publications

(25 citation statements)

References 36 publications

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“…Other studies show that the lifetime of lysozyme clusters is finite on millisecond time scales 77, 78 . The presented lifetimes are also not in contradiction with reports for solutions of bovine β-lactoglobulin (BLG) that indicate that BLG clusters are static on NSE observation time scale, i.e.…”

Section: Resultsmentioning

confidence: 98%

Slow-Down in Diffusion in Crowded Protein Solutions Correlates with Transient Cluster Formation

et al. 2017

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For a long time the effect of crowded cellular environment on protein dynamics has been largely ignored. Recent experiments indicate that proteins diffuse much slower in a living cell than in a diluted solution and further studies suggest that the diffusion depends on the local surrounding. Here, detailed insight into how diffusion depends on protein-protein contacts is presented based on extensive all-atom molecular dynamics simulations of concentrated villin head piece solutions. After force field adjustments in the form of increased protein-water interactions to reproduce experimental data, translational and rotational diffusion was analyzed in detail. While internal protein dynamics remained largely unaltered, rotational diffusion was found to slow down more significantly than translational diffusion as the protein concentration increased. The decrease in diffusion is interpreted in terms of a transient formation of protein clusters. These clusters persist on sub-microsecond time scales and follow distributions that increasingly shift toward larger cluster size with increasing protein concentrations. Weighting diffusion coefficients estimated for different clusters extracted from the simulations with the distribution of clusters largely reproduces the overall observed diffusion rates, suggesting that transient cluster formation is a primary cause for a slow-down in diffusion upon crowding with other proteins.

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Section: Resultsmentioning

confidence: 98%

Slow-Down in Diffusion in Crowded Protein Solutions Correlates with Transient Cluster Formation

et al. 2017

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“…We suggest two interpretations of these results. One is that the increase in the apparent volume of lysozyme was due to the formation of clusters . Cametti et al .…”

Section: Resultsmentioning

confidence: 99%

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy

2015

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We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5-300 mg ml(-1)). No changes in the amide-I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm(-1) and the ratios of the intensities of the Tyr bands at 856 and 837 cm(-1), the Trp bands at 870 and 877 cm(-1), and the bands at 2940 (CH stretching) and 3420 cm(-1) (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high-concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals.

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“…To find out the spectral signatures induced by crowded environment, lysozyme concentration dependence measurement of ANS fluorescence was carried out and the spectral differences between dilute and concentrated conditions were discussed. Another advantage using lysozyme as a model system is that the molecular behavior of concentered lysozyme solutions has been well investigated by various analytical methods; therefore, it could be possible to discuss the current results in detail and obtain further understanding by comparing to previous studies …”

Section: Resultsmentioning

confidence: 68%

“…The drastic increase in ANS fluorescence of lysozyme concentrated solutions over 50 mg/ml may indicate the excluded volume effect between molecules in the clusters. NMR studies compensate the solvation points of view, showing that the number of hydration waters decreases and only the external water layer is removed, while bounded water molecules can remain over 100 mg/ml solution . Although ANS fluorescence is not directly affected by the external hydration layer, the decrease of external hydration layer shortens the distance between lysozyme molecules, which may increase the excluded volume effect.…”

Section: Discussionmentioning

confidence: 91%

Spectroscopic Analysis of Protein‐Crowded Environments Using the Charge‐Transfer Fluorescence Probe 8‐Anilino‐1‐Naphthalenesulfonic Acid

Ota

Takano

2019

ChemPhysChem

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The molecular behaviors of proteins under crowding conditions are crucial for understanding the protein actions in intracellular environments. Under a crowded environment, the distance between protein molecules is almost the same size as the molecular level, thus, both the excluded volume effect and short ranged soft chemical interaction on protein surface could induce the complicated influence on the protein behavior cooperatively. Recently, various kinds of analytical approaches from macroscopic to microscopic aspects have been made to evaluate the crowding effect. The method, however, has not been established to evaluate the surface specific interactions on protein surface. In this study, the analytical method to evaluate the crowding effect has been suggested by using a charge‐transfer fluorescence probe, ANS. By employing the unique property of ANS attaching to charged residues on the surface of lysozyme, the crowding effect was focused, while the case was compared as a reference, in which ANS is confined in hydrophobic pockets of BSA. Consequently, the surface specific changes of fluorescence spectra were readily observed under the crowded environment, whereas the fluorescence spectra of ANS in protein inside did not change. This result suggests the fluorescence spectra of ANS binding to protein surface have the capability to estimate the crowding effect of proteins.

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NMR Detection of an Equilibrium Phase Consisting of Monomers and Clusters in Concentrated Lysozyme Solutions

Cited by 27 publications

References 36 publications

Slow-Down in Diffusion in Crowded Protein Solutions Correlates with Transient Cluster Formation

Slow-Down in Diffusion in Crowded Protein Solutions Correlates with Transient Cluster Formation

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy

Spectroscopic Analysis of Protein‐Crowded Environments Using the Charge‐Transfer Fluorescence Probe 8‐Anilino‐1‐Naphthalenesulfonic Acid

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