Lack of Dependence of the Sizes of the Mesoscopic Protein Clusters on Electrostatics

Vorontsova, Maria A.; Chan, Ho Yin; Lubchenko, Vassiliy; Vekilov, Peter G.

doi:10.1016/j.bpj.2015.09.025

Cited by 44 publications

(103 citation statements)

References 70 publications

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“…We determine the aggregates' radii from their Brownian trajectories, extracted from sequences of OIM images. 43,44 The aggregates exhibit a relatively narrow size distribution ( Figure 2c) with an average of R = 45 ± 5 nm at 2 µM and 15 o C. Both R and N are steady for at least two hours (Figure 2d,e), behaviors that stand in contrast to expectations for liquid-liquid separation, a first-order phase transition, 45,46 for which nucleation of new liquid droplets and their growth persist and R and N increase. 47,48 The reversibility of the observed aggregates is revealed by the concentration dependence of their number concentration ( Figure 2g) and the fraction of the solution volume that they occupy φ2 (Figure 2i).…”

Section: Mesoscopic Protein-rich Clusters In Solutions Of P53 R248qmentioning

confidence: 89%

“…Three behaviors of the p53 R248Q aggregates cohere with previous observations of mesoscopic protein-rich clusters of both globular proteins and wild type p53. 38,43,49,50 The average cluster radius R is, first, steady and, second, independent of the protein concentration C0. The third characteristic cluster behavior exhibited by the p53 R248 aggregates is the near-exponential increase of N and φ2 with C0 (Figure 2 h,j).…”

Section: Mesoscopic Protein-rich Clusters In Solutions Of P53 R248qmentioning

confidence: 97%

“…We monitored solutions of p53 R248Q with oblique illumination microcopy (OIM, Figure 2a). 43,44 This method records speckles of light scattered by individual solution inhomogeneities and is particularly suited to detection of mesoscopic aggregates since, according to the Rayleigh law, the scattered light intensity scales with the sixth power of the scatterers' size. The solutions were filtered through low-protein binding 220 nm filters to remove extrinsic inhomogeneities and loaded on the microscope within 10 -20 min of preparation.…”

Section: Mesoscopic Protein-rich Clusters In Solutions Of P53 R248qmentioning

confidence: 99%

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Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Yang

Saeedi

Davtyan

et al. 2020

Preprint

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The oncogenic properties of mutant p53 have been ascribed to destabilization of the p53 conformation, followed by aggregation into insoluble fibrils. Here we combine immunofluorescent 3D confocal microscopy of breast cancer cells expressing the p53 mutant Arg248Gln (R248Q) with light scattering from solutions of the purified protein and molecular simulations to probe the mechanisms that govern phase behaviors of the mutant across multiple length scales, from cellular to molecular. We establish that p53 R248Q forms mesoscopic proteinrich clusters, an anomalous liquid phase with several unique properties. We demonstrate that the clusters host and facilitate the nucleation of amyloid fibrils. The distinct characteristics of the clusters of R248Q and wild-type p53 and theoretical models indicate that p53 condensation into clusters is driven by the structural destabilization of the core domain and not by interactions of its extensive disordered region. Two-step nucleation of mutant p53 amyloids suggests means to IntroductionThe wild type p53 protein is a potent cancer suppressor, which is inactivated in almost every tumor, either through mutations in the TP53 gene (in 50% or more of human cancers) or deregulation of its associated pathways. 1-6 By contrast, p53 mutants emerge as potent cancer promoters because they exert a dominant-negative (DN) effect on the wild-type variant and also display oncogenic gain-of-function (GOF) properties by inhibiting other cancer suppressors. 1 Several mechanisms of cancer promotion by mutant p53 have been discussed. [6][7][8][9][10][11][12] It was recently suggested that the structural destabilization of the p53 mutants and the associated aggregation into insoluble, degradation resistant structures, designated fibrils, may play a decisive role in their oncogenicity. 2,[13][14][15] Concurrently, fibril suppression, for instance, by stabilization of the mutant p53 conformation, has been identified as a general way to fight cancer. 2,16 A parallel development identified a new form of biological organization, liquid-liquid phase separation, which constitutes several common membraneless organelles such as nucleoli, Cajal bodies, and P granules. [17][18][19][20][21] Many proteins forming dense liquid condensates in live cells incorporate significant disordered regions and condensation at the low concentrations of these proteins in the nucleoplasm and cytoplasm has been attributed to stronger intermolecular attraction due to the unstructured segments. 19,20 Furthermore, it is well-appreciated that protein dense liquids can serve as precursors and facilitators of higher order organization such as ordering into crystals 22-25 and the formation of polymers of sickle hemoglobin. 26,27 Theoretical analyses and recent experiments suggest that a similar precursor mechanism would apply to the nucleation of amyloid fibrils. [28][29][30][31][32][33]

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Section: Mesoscopic Protein-rich Clusters In Solutions Of P53 R248qmentioning

confidence: 89%

Section: Mesoscopic Protein-rich Clusters In Solutions Of P53 R248qmentioning

confidence: 97%

Section: Mesoscopic Protein-rich Clusters In Solutions Of P53 R248qmentioning

confidence: 99%

See 1 more Smart Citation

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Yang

Saeedi

Davtyan

et al. 2020

Preprint

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“…Their diameters are of order 100 nm (22,26,34,35) and each cluster contains 10 4 -10 5 protein molecules (26, 32-34, 36, 37). With lysozyme, a robust protein used as a model in several biophysics fields, cluster formation is reversible and the clusters exchange protein with the host solution (33,(37)(38)(39)(40). Remarkably, the responses of the characteristic cluster size and phase volume to variations of the ionic strength, pH, and additive concentration are decoupled and the cluster size is independent of protein concentration, solution ionic strength, and pH (39,40).…”

Section: Introductionmentioning

confidence: 99%

“…With lysozyme, a robust protein used as a model in several biophysics fields, cluster formation is reversible and the clusters exchange protein with the host solution (33,(37)(38)(39)(40). Remarkably, the responses of the characteristic cluster size and phase volume to variations of the ionic strength, pH, and additive concentration are decoupled and the cluster size is independent of protein concentration, solution ionic strength, and pH (39,40). These findings are in sharp contrast to observations for typical phase transitions, in which the size of the incipient domains and the volume occupied by the emerging phase increase or decrease concurrently (41).…”

Section: Introductionmentioning

confidence: 99%

Weakly-bound Dimers that Underlie the Crystal Nucleation Precursors in Lysozyme Solutions

Vekilov

McCabe

Angel

et al. 2018

Preprint

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Protein crystallization is central to understanding of molecular structure in biology, a vital part of processes in the pharmaceutical industry, and a crucial component of numerous disease pathologies. Crystallization starts with nucleation and how nucleation proceeds determines the crystallization rate and essential properties of the resulting crystal population. Recent results with several proteins indicate that crystals nucleate within preformed mesoscopic protein-rich clusters. The origin of the mesoscopic clusters is poorly understood. In the case of lysozyme, a common model of protein biophysics, earlier findings suggest that clusters exist owing to the dynamics of formation and decay of weakly-bound transient dimers. Here we present evidence of a weakly bound lysozyme dimer in solutions of this protein. We employ two electrospray mass spectrometry techniques, a combined ion mobility separation mass spectrometry and a high-resolution implementation. To enhance the weak but statistically-significant dimer signal we develop a method based on the residuals between the maxima of the isotope peaks in Fourier space and their Gaussian envelope. We demonstrate that these procedures sensitively detect the presence of a noncovalently bound dimer and distinguish its signal from other polypeptides, noise, and sampling artefacts. These findings contribute essential elements of the crystal nucleation mechanism of lysozyme and other proteins and suggest pathways to control nucleation and crystallization by enhancing or suppressing weak oligomerization.

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Rational Design of a Self‐Assembling High Performance Organic Nanofluorophore for Intraoperative NIR‐II Image‐Guided Tumor Resection of Oral Cancer

et al. 2023

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The first line of treatment for most solid tumors is surgical resection of the primary tumor with adequate negative margins. Incomplete tumor resections with positive margins account for over 75% of local recurrences and the development of distant metastases. In cases of oral cavity squamous cell carcinoma (OSCC), the rate of successful tumor removal with adequate margins is just 50–75%. Advanced real‐time imaging methods that improve the detection of tumor margins can help improve success rates,overall safety, and reduce the cost. Fluorescence imaging in the second near‐infrared (NIR‐II) window has the potential to revolutionize the field due to its high spatial resolution, low background signal, and deep tissue penetration properties, but NIR‐II dyes with adequate in vivo performance and safety profiles are scarce. A novel NIR‐II fluorophore, XW‐03‐66, with a fluorescence quantum yield (QY) of 6.0% in aqueous media is reported. XW‐03‐66 self‐assembles into nanoparticles (≈80 nm) and has a systemic circulation half‐life (t1/2) of 11.3 h. In mouse models of human papillomavirus (HPV)+ and HPV‐ OSCC, XW‐03‐66 outperformed indocyanine green (ICG), a clinically available NIR dye, and enabled intraoperative NIR‐II image‐guided resection of the tumor and adjacent draining lymph node with negative margins. In vitro and in vivo toxicity assessments revealed minimal safety concerns for in vivo applications.

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Lack of Dependence of the Sizes of the Mesoscopic Protein Clusters on Electrostatics

Cited by 44 publications

References 70 publications

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Weakly-bound Dimers that Underlie the Crystal Nucleation Precursors in Lysozyme Solutions

Rational Design of a Self‐Assembling High Performance Organic Nanofluorophore for Intraoperative NIR‐II Image‐Guided Tumor Resection of Oral Cancer

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