Adsorbed Protein Secondary and Tertiary Structures by Circular Dichroism and Infrared Spectroscopy with Refractive Index Matched Emulsions

Husband, Fiona A.; Garrood, Martin; Mackie, Alan; Burnett, Gary R.; Wilde, Peter J.

doi:10.1021/jf000688z

Cited by 110 publications

(90 citation statements)

References 28 publications

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“…What is the nature of the energy barrier against adsorption to the interface? To study the conformation of BslA in aqueous solution and at an oil-water interface, circular dichroism (CD) spectroscopy of WT-BslA was performed in aqueous solution and in refractive index matched emulsions (23). Refractive index matching enables the generation of oil-in-water emulsions without the light scattering that interferes with spectroscopic measurements.…”

Section: Bsla Undergoes a Conformational Change To A Structure Enrichmentioning

confidence: 99%

Interfacial self-assembly of a bacterial hydrophobin

Bromley

Morris

Hobley

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

132

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The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surfaceexposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded β-sheet. This form then self-assembles into a wellordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.BslA | interfacial self-assembly | bacterial hydrophobin | Bacillus subtilis | biofilm

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Section: Bsla Undergoes a Conformational Change To A Structure Enrichmentioning

confidence: 99%

Interfacial self-assembly of a bacterial hydrophobin

Bromley

Morris

Hobley

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

132

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“…Recently, however, CD spectropolarimetry of refractive-index matched emulsions (RIMEs) has been used to investigate the secondary structure of adsorbed proteins (8,9). A RIME is prepared by the addition of glycerol to the aqueous, continuous phase of the o/w emulsion, raising the refractive index of the water to that of the oil and rendering the emulsion transparent.…”

Section: Secondary Structure Of Adsorbed Proteinmentioning

confidence: 99%

Characterization of the Emulsification Properties of 2S Albumins from Sunflower Seed

Burnett

Rigby

Mills

et al. 2002

Journal of Colloid and Interface Science

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“…More recently also external reflection circular dichroism has been used to assess this information [33]. From a combination of these techniques, several authors conclude that only limited changes in the conformation of proteins occurs upon adsorption at the air-water interface [28,32,34,[36][37][38][39][40]. Maximum changes of up to 10% are observed in the secondary structure, but the globular folding state of the protein is generally found to remain intact [34].…”

Section: Introductionmentioning

confidence: 99%

The adsorption and unfolding kinetics determines the folding state of proteins at the air–water interface and thereby the equation of state

Wierenga¹,

Egmond²,

Voragen³

et al. 2006

Journal of Colloid and Interface Science

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Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For β-lactoglobulin the adsorbed amount (Γ ) needed to reach a certain surface pressure (Π) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Π-Γ , and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).

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Adsorbed Protein Secondary and Tertiary Structures by Circular Dichroism and Infrared Spectroscopy with Refractive Index Matched Emulsions

Cited by 110 publications

References 28 publications

Interfacial self-assembly of a bacterial hydrophobin

Interfacial self-assembly of a bacterial hydrophobin

Characterization of the Emulsification Properties of 2S Albumins from Sunflower Seed

The adsorption and unfolding kinetics determines the folding state of proteins at the air–water interface and thereby the equation of state

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