Spackling the Crack: Stabilizing Human Fibroblast Growth Factor-1 by Targeting the N and C terminus β-Strand Interactions

Dubey, Vikash Kumar; Lee, Jihun; Somasundaram, Thayumana; Blaber, Sachiko I.; Blaber, Michael

doi:10.1016/j.jmb.2007.05.065

Cited by 29 publications

(40 citation statements)

References 53 publications

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“…One must decouple the effects of the two to control folding (9) and design fast folding scaffolds. These scaffolds can then be programmed for specific function (10).…”

mentioning

confidence: 99%

Extracting function from a β-trefoil folding motif

Gosavi

Whitford

Jennings

et al. 2008

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

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Despite having remarkably similar three-dimensional structures and stabilities, IL-1β promotes signaling, whereas IL-1Ra inhibits it. Their energy landscapes are similar and have coevolved to facilitate competitive binding to the IL-1 receptor. Nevertheless, we find that IL-1Ra folds faster than IL-1β. A structural alignment of the proteins shows differences mainly in two loops, a β-bulge of IL-1β and a loop in IL-1Ra that interacts with residue K145 and connects β-strands 11 and 12. Bioassays indicate that inserting the β-bulge from IL-1β confers partial signaling capability onto a K145D mutant of IL-1Ra. Based on the alignment, mutational assays and our computational folding results, we hypothesize that functional regions are not central to the β-trefoil motif and cause slow folding. The IL-1β β-bulge facilitates activity and replacing it by the IL-1Ra β-turn results in a hybrid protein that folds faster than IL-1β. Inserting the β11–β12 connecting-loop, which aids inhibition, into either IL-1β or the hybrid protein slows folding. Thus, regions that aid function (either through activity or inhibition) can be inferred from folding traps via structural differences. Mapping functional properties onto the numerous folds determined in structural genomics efforts is an area of intense interest. Our studies provide a systematic approach to mapping the functional genomics of a fold family.

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“…One must decouple the effects of the two to control folding (9) and design fast folding scaffolds. These scaffolds can then be programmed for specific function (10).…”

mentioning

confidence: 99%

Extracting function from a β-trefoil folding motif

Gosavi

Whitford

Jennings

et al. 2008

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

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“…Mutations were combined with a screen for protein folding, stability, and solubility, and were retained if they were neutral or improved upon such properties. Maximum use was made of previously published data (26)(27)(28)(29)(30)(31)(32)(33)(34) as well as sequence analysis to identify useful symmetric mutations in FGF-1. A summary of the application of specific transforms in pursuing a symmetric deconstruction of FGF-1 follows.…”

Section: Methodsmentioning

confidence: 99%

“…The SYM7ΔΔ mutant was initially modified by the inclusion of two previously described stabilizing point mutations (Lys12Val and Pro134Val) (30) to produce the SYM9ΔΔ mutant. These mutations are located in the adjacent first and last β-strands of the β-barrel of the architecture, respectively, and represent a "discontinuous" β-turn within the threefold architecture.…”

Section: Methodsmentioning

confidence: 99%

Experimental support for the evolution of symmetric protein architecture from a simple peptide motif

Lee

Blaber²

2010

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

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140

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The majority of protein architectures exhibit elements of structural symmetry, and "gene duplication and fusion" is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique "top-down symmetric deconstruction" strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed "conserved architecture" model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes.protein design | protein folding | protein symmetry

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“…The mutant proteins were developed via a series of protein stability and folding studies with FGF-1 and were identified experimentally as having enhanced stability, functional half-life, or mitogenic activity. 6,[22][23][24][25][26][27][28] The previously described thermodynamic stability properties and available in vitro mitogenic activity as well as in vivo half-life values are summarized in Table I. The rationale used for the selection of mutants examined in this study is based on the results in Table I (WT FGF-1 and mutants are labeled A-L).…”

Section: Selection Of Fgf-1 Mutantsmentioning

confidence: 99%

“…29 Alternatively, the P134V/C117V (E) mutant has enhanced stability (DDG ¼ À9.3 kJ/mol) yet exhibits similar mitogenic activity in comparison to WT FGF-1 (A). Other examples include K12V/C117V (F) and K12V/C117V/ P134V (H) which are stabilizing mutations that, in the absence of heparin, achieve similar mitogenic potency to that of WT formulated in the presence of heparin (mutants E, F, and H were designed to stabilize N-and C-termini b-strand interactions, a region of known structural weakness within FGF-1 24 ) The mutants C83T/C117V/K12V (D), C83T/ C117V/L44F/F132W (I), and oxidized A66C (G) have eliminated one or more free thiols, in comparison to WT FGF-1 (A). These mutations substantially increase their in vitro functional half-life with varying effects on thermostability (by mutating exclusively at solvent-inaccessible positions, thereby limiting immunogenic potential 6,27 ).…”

Section: Selection Of Fgf-1 Mutantsmentioning

confidence: 99%

An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1

et al. 2012

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Acidic fibroblast growth factor-1 (FGF-1) is an angiogenic protein which requires binding to a polyanion such as heparin for its mitogenic activity and physicochemical stability. To evaluate the extent to which this heparin dependence on solution stability could be reduced or eliminated, the structural integrity and conformational stability of 10 selected FGF-1 mutants were examined as a function of solution pH and temperature by a series of spectroscopic methods including circular dichroism, intrinsic and extrinsic fluorescence spectroscopy and static light scattering. The biophysical data were summarized in the form of colored empirical phase diagrams (EPDs). FGF-1 mutants were identified with stability profiles in the absence of heparin comparable to that of wild-type FGF-1 in the presence of heparin while still retaining their biological activity. In addition, a revised version of the EPD methodology was found to provide an information rich, high throughput approach to compare the effects of mutations on the overall conformational stability of proteins in terms of their response to environmental stresses such as pH and temperature.

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Spackling the Crack: Stabilizing Human Fibroblast Growth Factor-1 by Targeting the N and C terminus β-Strand Interactions

Cited by 29 publications

References 53 publications

Extracting function from a β-trefoil folding motif

Extracting function from a β-trefoil folding motif

Experimental support for the evolution of symmetric protein architecture from a simple peptide motif

An empirical phase diagram approach to investigate conformational stability of “second‐generation” functional mutants of acidic fibroblast growth factor‐1

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