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A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion.

ABSTRACT: Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions.

SUBMITTER: Hansen CL

PROVIDER: S-EPMC139178 | biostudies-literature | 2002 Dec

REPOSITORIES: biostudies-literature

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A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion.

Hansen Carl L CL Skordalakes Emmanuel E Berger James M JM Quake Stephen R SR

Proceedings of the National Academy of Sciences of the United States of America 20021216 26

Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active va ...[more]

PMID: 12486223

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Project description:BackgroundInterleukin 4 (IL-4) is a key regulator of the immune system and an important factor in the development of allergic hypersensitivity. Together with interleukin 13 (IL-13), IL-4 plays an important role in exacerbating allergic and asthmatic symptoms. For signal transduction, both cytokines can utilise the same receptor, consisting of the IL-4Ralpha and the IL-13Ralpha1 chain, offering an explanation for their overlapping biological functions. Since both cytokine ligands share only moderate similarity on the amino acid sequence level, molecular recognition of the ligands by both receptor subunits is of great interest. IL-4 and IL-13 are interesting targets for allergy and asthma therapies. Knowledge of the binding mechanism will be important for the generation of either IL-4 or IL-13 specific drugs.ResultsWe present a structure/function analysis of the IL-4 ligand-receptor interaction. Structural determination of a number of IL-4 variants together with in vitro binding studies show that IL-4 and its high-affinity receptor subunit IL-4Ralpha interact via a modular protein-protein interface consisting of three independently-acting interaction clusters. For high-affinity binding of wild-type IL-4 to its receptor IL-4Ralpha, only two of these clusters (i.e. cluster 1 centered around Glu9 and cluster 2 around Arg88) contribute significantly to the free binding energy. Mutating residues Thr13 or Phe82 located in cluster 3 to aspartate results in super-agonistic IL-4 variants. All three clusters are fully engaged in these variants, generating a three-fold higher binding affinity for IL-4Ralpha. Mutagenesis studies reveal that IL-13 utilizes the same main binding determinants, i.e. Glu11 (cluster 1) and Arg64 (cluster 2), suggesting that IL-13 also uses this modular protein interface architecture.ConclusionThe modular architecture of the IL-4-IL-4Ralpha interface suggests a possible mechanism by which proteins might be able to generate binding affinity and specificity independently. So far, affinity and specificity are often considered to co-vary, i.e. high specificity requires high affinity and vice versa. Although the binding affinities of IL-4 and IL-13 to IL-4Ralpha differ by a factor of more than 1000, the specificity remains high because the receptor subunit IL-4Ralpha binds exclusively to IL-4 and IL-13. An interface formed by several interaction clusters/binding hot-spots allows for a broad range of affinities by selecting how many of these interaction clusters will contribute to the overall binding free energy. Understanding how proteins generate affinity and specificity is essential as more and more growth factor receptor families show promiscuous binding to their respective ligands. This limited specificity is, however, not accompanied by low binding affinities.

Dataset Information

A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion.

Publications

A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion.

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