Competitive multicomponent anion exchange adsorption of proteins at the single molecule level

Kisley, Lydia; Patil, Ujwal; Dhamane, Sagar; Kourentzi, Katerina; Tauzin, Lawrence J.; Willson, Richard C.; Landes, Christy F.

doi:10.1039/c7an00701a

Cited by 18 publications

(12 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Additionally, circular dichroism (CD) spectroscopy is utilized to quantify structural changes of proteins at the stationary phase support and relate to TIRF and FPLC results. TIRF single-molecule tracking has been recently shown to reveal interfacial protein dynamics on a range of surfaces, including natural and synthetic polymers (44)(45)(46)(47)(48), and is ideal for measuring interfacial dynamics as it intrinsically suppresses background signal from emitters diffusing in the bulk solution. TIRF microscopy has been applied in many other systems including nanoparticle catalysis (49)(50)(51), DNA hybridization kinetics (52)(53)(54), and protein transport in live cells (54,55).…”

Section: Significancementioning

confidence: 99%

A mechanistic examination of salting out in protein–polymer membrane interactions

Moringo

Bishop

Shen

et al. 2019

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

Self Cite

View full text Add to dashboard Cite

Developing a mechanistic understanding of protein dynamics and conformational changes at polymer interfaces is critical for a range of processes including industrial protein separations. Salting out is one example of a procedure that is ubiquitous in protein separations yet is optimized empirically because there is no mechanistic description of the underlying interactions that would allow predictive modeling. Here, we investigate peak narrowing in a model transferrin–nylon system under salting out conditions using a combination of single-molecule tracking and ensemble separations. Distinct surface transport modes and protein conformational changes at the negatively charged nylon interface are quantified as a function of salt concentration. Single-molecule kinetics relate macroscale improvements in chromatographic peak broadening with microscale distributions of surface interaction mechanisms such as continuous-time random walks and simple adsorption–desorption. Monte Carlo simulations underpinned by the stochastic theory of chromatography are performed using kinetic data extracted from single-molecule observations. Simulations agree with experiment, revealing a decrease in peak broadening as the salt concentration increases. The results suggest that chemical modifications to membranes that decrease the probability of surface random walks could reduce peak broadening in full-scale protein separations. More broadly, this work represents a proof of concept for combining single-molecule experiments and a mechanistic theory to improve costly and time-consuming empirical methods of optimization.

show abstract

Section: Significancementioning

confidence: 99%

A mechanistic examination of salting out in protein–polymer membrane interactions

Moringo

Bishop

Shen

et al. 2019

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…The interactions of proteins with surfaces have implications in biocompatibility of a surface, − protein separation, − and pharmaceutical nanoparticle development . Protein adsorption affects the ability of both the protein and the surface to fulfill their intended purpose in these capacities .…”

Section: Introductionmentioning

confidence: 99%

Power Law Behavior in Protein Desorption Kinetics Originating from Sequential Binding and Unbinding

et al. 2020

View full text Add to dashboard Cite

The study of protein adsorption at the single molecule level has recently revealed that the adsorption is reversible, but with a long-tailed residence time distribution which can be approximated with a sum of exponential functions putatively related to distinct adsorption sites. Here it is proposed that the shape of the residence time distribution results from an adsorption process with sequential and reversible steps that contribute to overall binding strength resembling “zippering”. In this model, the survival function of the residence time distribution of single proteins varies from an exponential distribution for a single adsorption step to a power law distribution with exponent −1/2 for a large number of adsorption steps. The adsorption of fluorescently labeled fibrinogen to glass surfaces is experimentally studied with single molecule imaging. The experimental residence time distribution can be readily fit by the proposed model. This demonstrates that the observed long residence times can arise from stepwise adsorption rather than rare but strong binding sites and provides guidance for the control of protein adsorption to biomaterials.

show abstract

“…In reality, all separations are performed in a complex environment with multiple analytes competing for adsorption sites. While we have previously studied a multicomponent sample, only one analyte was labeled, requiring the dynamics of the other unlabeled analyte to be inferred . If labels were placed on each analyte in future work, their dynamics could be directly imaged to answer questions such as: are some binding sites preferred by one analyte compared to the other?…”

Section: Single-molecule Imaging and Bottom-up Separationsmentioning

confidence: 99%

“…Microfluidics and solvent mixing could be incorporated to monitor dynamics that occur during gradient elutions. Higher concentrations of analytes could be studied by mixing labeled and unlabeled molecules , or utilizing unique sample hardware geometries that limit the excitation to a small volume. , High-concentration samples could observe thermodynamic tailing caused by overloading, in addition to the established studies of kinetic tailing at low concentrations . Further complex conditions include incorporating high pressures, temperatures, and oxidative/corrosive chemicals that are used in more extreme separations such as (ultra) high-performance liquid chromatography.…”

Section: Studying Complex Single-molecule Samples More Relevant To Re...mentioning

confidence: 99%

“…While we have previously studied a multicomponent sample, only one analyte was labeled, requiring the dynamics of the other unlabeled analyte to be inferred. 41 If labels were placed on each analyte in future work, their dynamics could be directly imaged to answer questions such as: are some binding sites preferred by one analyte compared to the other? How do the kinetics differ at the same binding site with different analytes?…”

mentioning

confidence: 99%

See 1 more Smart Citation

Transforming Separation Science with Single-Molecule Methods

et al. 2020

Self Cite

View full text Add to dashboard Cite

Competitive multicomponent anion exchange adsorption of proteins at the single molecule level

Cited by 18 publications

References 33 publications

A mechanistic examination of salting out in protein–polymer membrane interactions

A mechanistic examination of salting out in protein–polymer membrane interactions

Power Law Behavior in Protein Desorption Kinetics Originating from Sequential Binding and Unbinding

Transforming Separation Science with Single-Molecule Methods

Contact Info

Product

Resources

About