Engineering a novel, stable dimeric streptavidin with lower isoelectric point

Aslan, Filiz M.; Yu, Yanke; Vajda, Sándor; Mohr, Scott C.; Cantor, Charles R.

doi:10.1016/j.jbiotec.2006.08.014

Cited by 17 publications

(15 citation statements)

References 27 publications

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“…The remaining protein layers all produced slightly negative zeta potential measurements, consistent with their respective isoelectric points. 27,28 Collectively, this evidence strongly suggests that the layers were successfully tethered. It is difficult to decipher from the SEM images whether there is a difference in the thickness or surface morphology of the microcapsules as the layers are added to the surface.…”

Section: ■ Discussionmentioning

confidence: 78%

Biofunctionalization of PEGylated Microcapsules for Exclusive Binding to Protein Substrates

et al. 2014

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Targeted delivery of drugs to specific diseased sites in the body is an area of research that has attracted many studies, particularly in drug deliveries that utilize microparticles. By achieving targeted delivery of a drug, one can increase the efficacy of the treatment, thus, reducing unwanted side effects. This study aims to synthesize microcapsules that are able to target and adsorb to specific proteins (i.e., collagen type IV and fibronectin) through antibody-antigen interactions, while simultaneously suppressing any unspecific binding, a characteristic that is commonly observed in polyelectrolyte microcapsule-protein interactions. This is accomplished by creating an antibody-functionalized poly(ethylene glycol) (PEG) assembly within the microcapsule structure. Site-specific adsorption of these microcapsules is tested using protein micropatterns. Results show that significant adsorption is achieved on the target protein, with unspecific adsorptions being heavily suppressed on control proteins. In conclusion, we have successfully manufactured microcapsules that specifically and exclusively bind to their complementary target area. This paves the way for future in vivo experiments using microcapsules as targeted drug carriers.

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Section: ■ Discussionmentioning

confidence: 78%

Biofunctionalization of PEGylated Microcapsules for Exclusive Binding to Protein Substrates

et al. 2014

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“…One of these is the longstanding question regarding cause/effect in the coupled events of D128-biotin and bond stretching and water entry into the binding pocket to hydrate D128. We establish that at least in the current dimeric system (which itself has been the subject of previous investigations 21 ), it is the bond stretching which forms the bottleneck in the early parts of the unbinding. Our reported residence times for the dimeric system are in rough agreement with what one expects after taking into account the difference in binding affinities between the dimeric and the tetrameric systems.…”

Section: Discussionmentioning

confidence: 56%

Molecular determinants and bottlenecks in the unbinding dynamics of biotin-streptavidin

Tiwary

2017

Preprint

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Biotin-streptavidin is a very popular system used to gain insight into protein-ligand interactions. In its tetrameric form, it is well-known for its extremely long residence times, being one of the strongest known non-covalent interactions in nature, and is heavily used across the biotechnological industry. In this work we gain understanding into the molecular determinants and bottlenecks in the unbinding of the dimeric biotinstreptavidin system in its wild type and with N23A mutation. Using new enhanced sampling methods with full atomistic resolution, we reproduce the variation caused by N23A mutation in experimentally reported residence time. We also answer a longstanding question regarding cause/effect in the coupled events of bond stretching and bond hydration during unbinding and establish that in this system, it is the bond stretching and not hydration which forms the bottleneck in the early parts of the unbinding. We believe these calculations represent a step forward in the use of atomistic simulations to study pharmacodynamics. An improved understanding of biotin-streptavidin unbinding dynamics should also have direct benefits in biotechnological and nanobiotechnological applications.The interaction of streptavidin with biotin in the homotetramer form is one of the strongest known noncovalent interactions in nature. 1-4 This system forms a central component of countless biological, biotechnological and nano-biotechnological applications, and has long been used as a model system to understand the molecular basis of high affinity binding. 5,6 Designing specific mutations in streptavidin allows tailoring a wide range of affinities and rate constants 7 as needed for diverse practical applications. Further, given that many of the structural motifs found in biotin-streptavidin are utilized in numerous other protein-ligand interactions, it is very desirable to map out the structural and dynamical motifs underlying the slow dissociation kinetics in this system. Experiments have reported both the binding affinity (K D ) and the binding/unbinding rate constants (k on , k of f respectively, with K D = k on /k of f ) for this system with and without mutations in the host. Of these numbers, the unbinding rate constant k of f (also often represented through its inverse, the residence time) has recently received a lot of attention as possibly being a much better predictor of drug efficacy compared to the static K D . 8-10 While experiments can provide a direct measurement of k of f , it is not so easy to derive from experiments direct information into the molecular determinants of the residence time. These determinants could come from a diverse range -protein-ligand contacts, solvation, protein/ligand flexibilities and many others. Having an atomistic resolution understanding of the role played by these determinants in the unbinding dynamics, one could then propose structural modifications that would assist in rational design of drugs with desired residence times. With the advent of petaflop computing and reliable forc...

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“…2c; Lim et al, 2011). The charged residues also improve the solubility of the molecule, which is a frequent problem during streptavidin engineering (Aslan et al, 2007; Wu and Wong, 2005). Additionally, we mutated L109, L124, V125, and H127 to their rhizavidin counterparts, N103, E115, Q116, and Q118, to prevent hydrophobic and aromatic interactions that contribute to the tetramer formation.…”

Section: Resultsmentioning

confidence: 99%

“…Most of the dimers and monomers that have been engineered to date are unstable, aggregation prone, and have low biotin affinity (Aslan et al, 2007; Laitinen et al, 2003; Sano et al, 1997; Wu and Wong, 2005). The limited affinity in designed monomers is attributed to the missing “hydrophobic lid” resulting from lost subunit interaction (Chilkoti et al, 1995; Sano and Cantor, 1995).…”

Section: Introductionmentioning

confidence: 99%

Novel heat resistant methyl‐tri(phenylethynyl)silane resin: Synthesis, characterization and thermal properties

Zhou

Feng

et al. 2006

J of Applied Polymer Sci

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Methyl-tri(phenylethynyl)silane (MTPES) was successfully synthesized by the reaction of lithium phenylacetylide with methyltrichlorosilane. The structure was characterized by HRMS, FTIR, Si-NMR, and elementary analysis. Thermal cure process was monitored by DSC, DMA, and FTIR. MTPES was heated to free flowing liquid around 1308C and thermally polymerized at 327-3778C to form thermoset. Thermal and oxidative properties were evaluated by TGA analysis. Thermoset exhibits extremely high heat-resistance and TGA curve in nitrogen shows the temperature of 5% weight loss (Td 5 ) of 6958C and total weight loss at 8008C of 7.1%. TGA shows a high Td 5 of 5658C even in air, although the total weight loss at 8008C was 56.1% of the initial weight, much higher than that in nitrogen. The high heat resistance of MTPES was ascribed to crosslinking reaction concerning ethynyl groups. Aging studies performed at elevated temperatures in air on a thermoset showed that MTPES is oxidatively stable to 3008C.

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Engineering a novel, stable dimeric streptavidin with lower isoelectric point

Cited by 17 publications

References 27 publications

Biofunctionalization of PEGylated Microcapsules for Exclusive Binding to Protein Substrates

Biofunctionalization of PEGylated Microcapsules for Exclusive Binding to Protein Substrates

Molecular determinants and bottlenecks in the unbinding dynamics of biotin-streptavidin

Novel heat resistant methyl‐tri(phenylethynyl)silane resin: Synthesis, characterization and thermal properties

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