Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Gast, Klaus; Nöppert, Angela; Müller-Frohne, Marlies; Zirwer, Dietrich; Damaschun, G.

doi:10.1007/s002490050033

Cited by 31 publications

(11 citation statements)

References 34 publications

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“…Taking into account the size of the fluorescence changes during refolding, we estimate that at 1000 sec the folding reaction is <3%–4% away from equilibrium in all the mutants of our model system. This is in agreement with earlier findings (Betton et al 1992; Missiakas et al 1992; Otto et al 1994; Gast et al 1997).…”

Section: Resultssupporting

confidence: 93%

Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase

Osváth¹,

Köhler²,

Závodszky³

et al. 2005

Protein Science

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The aim of this work is to shed more light on the effect of domain-domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single-tryptophan variants of the two-domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two-state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain-domain interactions direct the folding of both domains, but in an asymmetric way.

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Section: Resultssupporting

confidence: 93%

Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase

Osváth¹,

Köhler²,

Závodszky³

et al. 2005

Protein Science

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“…Simultaneous static and dynamic light scattering measurements were done with the same instrument at a scattering angle of 90°. The custom-built apparatus, equipped with a diode-pumped continuous wave laser (Millennia IIs, Spectra-Physics) and a high quantum yield avalanche photodiode, has been described in detail ( 27 ). Instead of using a commercially available stopped-flow mixing device ( 27 ), manual mixing (dead time about 10 s) was preferred in the present work.…”

Section: Methodsmentioning

confidence: 99%

“…The custom-built apparatus, equipped with a diode-pumped continuous wave laser (Millennia IIs, Spectra-Physics) and a high quantum yield avalanche photodiode, has been described in detail ( 27 ). Instead of using a commercially available stopped-flow mixing device ( 27 ), manual mixing (dead time about 10 s) was preferred in the present work. Manual mixing ensures stable solution composition, particularly during long-term experiments.…”

Section: Methodsmentioning

confidence: 99%

Rapid-Acting and Human Insulins: Hexamer Dissociation Kinetics upon Dilution of the Pharmaceutical Formulation

et al. 2017

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PurposeComparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions.MethodsDissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism.ResultsGlulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs.ConclusionKnowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-017-2233-0) contains supplementary material, which is available to authorized users.

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“…These were performed simultaneously at a scattering angle of 90°with a laboratory-built apparatus, presently equipped with a diode-pumped, continuous-wave laser Millennia IIs (Spectra-Physics, Darmstadt, Germany) and a high-quantum-yield avalanche photodiode as described elsewhere [21]. The translational diffusion coefficient D was obtained from the measured autocorrelation functions using the program CONTIN [22].…”

Section: Fluorescence Resonance Energy Transfer Analysismentioning

confidence: 99%

On the self-association potential of transmembrane tight junction proteins

Blasig

Winkler

Lassowski

et al. 2006

Cell. Mol. Life Sci.

Self Cite

118

120

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Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

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Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Cited by 31 publications

References 34 publications

Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase

Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase

Rapid-Acting and Human Insulins: Hexamer Dissociation Kinetics upon Dilution of the Pharmaceutical Formulation

On the self-association potential of transmembrane tight junction proteins

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