Stabilization of a Fibronectin Type III Domain by the Removal of Unfavorable Electrostatic Interactions on the Protein Surface

Koide, Akiko; Jordan, Michael R.; Horner, Scott R.; Batori, Vincent; Koide, Shohei

doi:10.1021/bi010916y

Cited by 66 publications

(98 citation statements)

References 37 publications

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“…However, Cota et al (2000) demonstrated that mutation of Pro5 to alanine does not effect stability, as would be expected if it does not contribute to the hydrophobic core of 10FnIII alone. Also, Asp7 makes unfavorable electrostatic interactions with other negatively charged residues on the surface of 10FnIII (Koide et al 2001), further validating the removal of the unstructured residues.…”

Section: Discussionmentioning

confidence: 75%

Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain

Olson

Roberts

2007

Protein Science

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Protein libraries based on natural scaffolds enable the generation of novel molecular tools and potential therapeutics by directed evolution. Here, we report the design and construction of a high complexity library (30 3 10 13 sequences) based on the 10th fibronectin type III domain of human fibronectin (10FnIII). We examined the bacterial expression characteristics and stability of this library using a green fluorescent protein (GFP)-reporter screen, SDS-PAGE analysis, and chemical denaturation, respectively. The high throughput GFP reporter screen demonstrates that a large fraction of our library expresses significant levels of soluble protein in bacteria. However, SDS-PAGE analysis of expression cultures indicates the ratio of soluble to insoluble protein expressed varies greatly for randomly chosen library members. We also tested the stabilities of several representative variants by guanidinium chloride denaturation. All variants tested displayed cooperative unfolding transitions similar to wild-type, and two exhibited free energies of unfolding equal to wild-type 10FnIII. This work demonstrates the utility of GFP-based screening as a tool for analysis of high-complexity protein libraries. Our results indicate that a vast amount of protein sequence space surrounding the 10FnIII scaffold is accessible for the generation of novel functions by directed as well as natural evolution.

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Section: Discussionmentioning

confidence: 75%

Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain

Olson

Roberts

2007

Protein Science

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“…A synthetic DNA fragment that encodes signal sequence of DsbA (17) was fused to the gene for the template, and the fusion gene was cloned into the phage display vector pAS38 (9). A stabilizing mutation (D7K) was also introduced (18).…”

Section: Methodsmentioning

confidence: 99%

High-affinity single-domain binding proteins with a binary-code interface

Koide

Gilbreth

Esaki

et al. 2007

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

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168

178

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High degrees of sequence and conformation complexity found in natural protein interaction interfaces are generally considered essential for achieving tight and specific interactions. However, it has been demonstrated that specific antibodies can be built by using an interface with a binary code consisting of only Tyr and Ser. This surprising result might be attributed to yet undefined properties of the antibody scaffold that uniquely enhance its capacity for target binding. In this work we tested the generality of the binary-code interface by engineering binding proteins based on a single-domain scaffold. We show that Tyr/Ser binary-code interfaces consisting of only 15-20 positions within a fibronectin type III domain (FN3; 95 residues) are capable of producing specific binding proteins (termed ''monobodies'') with a low-nanomolar Kd. A 2.35-Å x-ray crystal structure of a monobody in complex with its target, maltose-binding protein, and mutation analysis revealed dominant contributions of Tyr residues to binding as well as striking molecular mimicry of a maltose-binding protein substrate, ␤-cyclodextrin, by the Tyr/Ser binary interface. This work suggests that an interaction interface with low chemical diversity but with significant conformational diversity is generally sufficient for tight and specific molecular recognition, providing fundamental insights into factors governing protein-protein interactions.3D domain swapping ͉ antibody engineering ͉ combinatorial library ͉ phage display ͉ sequence diversity

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“…The phage display vectors contained the DsbA signal sequence (31), the circularly permutated Erbin PDZ domain, and the FN3 domain (22), in which the PDZ and FN3 domains were connected with a GGSGG linker. A stabilization mutation, D7N (32), was also introduced to FN3. Combinatorial libraries in which the BC, DE, and FG loops of FN3 were diversified were constructed by using Kunkel mutagenesis as previously described (22,33,34).…”

Section: Construction Of Phage Display Vectors and Combinatorial Librmentioning

confidence: 99%

Design of protein function leaps by directed domain interface evolution

Huang

Koide

Makabe

et al. 2008

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

Self Cite

107

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Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting ''affinity clamp'' had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution.affinity reagent ͉ epitope ͉ molecular evolution ͉ protein design ͉ PDZ domain D irected evolution-based protein engineering creates new protein functions by exploiting processes that occur during natural evolution of proteins. Protein evolution progresses via point mutations, duplication, and recombination of genes under selective pressure. The processes of gene duplication and subsequent sequence divergence (1) have been successfully recapitulated in directed evolution and computational protein design (2, 3) where preexisting active sites within natural protein scaffolds are altered to produce new functions (Fig.

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Stabilization of a Fibronectin Type III Domain by the Removal of Unfavorable Electrostatic Interactions on the Protein Surface

Cited by 66 publications

References 37 publications

Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain

Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain

High-affinity single-domain binding proteins with a binary-code interface

Design of protein function leaps by directed domain interface evolution

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