Control of protein functional dynamics by peptide linkers

Wriggers, Willy; Chakravarty, S.; Jennings, Patricia A.

doi:10.1002/bip.20291

Cited by 169 publications

(164 citation statements)

References 107 publications

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“…Thus, the screening method is not exact, possibly due to a dependence of proteolytic susceptibility on local structure (Parsell and Sauer, 1989;Wriggers et al, 2005). Nevertheless, it is shown in this work to provide a novel and potentially useful strategy for linker design.…”

Section: Discussionmentioning

confidence: 89%

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Kavoosi

Creagh

Kilburn

et al. 2007

Biotech & Bioengineering

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The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPS to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS-designed S(3)N(10) linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor X(a) processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S(3)N(10) linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the T(m) of GFP but did result in a destabilization, as seen by both a decrease in T(m) and DeltaH(cal), of CBM9. The degree of destabilization increased with decreasing length of the (PT)(x)P linker such that DeltaT(m) = -8.4 degrees C for the single P linker.

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Section: Discussionmentioning

confidence: 89%

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Kavoosi

Creagh

Kilburn

et al. 2007

Biotech & Bioengineering

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“…This fusion protein mimics natural bifunctional enzymes, such as Arabidopsis lysine-ketoglutarate reductase/saccharopine dehydrogenase (50), human 5-amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (3), Abies grandis abietadiene synthase (31), and lysine-ketoglutarate reductase/saccharopine dehydrogenase from developing soybean seeds (27). We joined Idi and NudB with a peptide linker because linkers can control favorable and unfavorable interactions between adjacent protein domains (47).…”

Section: Resultsmentioning

confidence: 99%

Synthetic Pathway for Production of Five-Carbon Alcohols from Isopentenyl Diphosphate

Chou

Keasling

2012

Appl Environ Microbiol

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f Synthetic biological pathways could enhance the development of novel processes to produce chemicals from renewable resources. On the basis of models that describe the evolution of metabolic pathways and enzymes in nature, we developed a framework to rationally identify enzymes able to catalyze reactions on new substrates that overcomes one of the major bottlenecks in the assembly of a synthetic biological pathway. We verified the framework by implementing a pathway with two novel enzymatic reactions to convert isopentenyl diphosphate into 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methylbutanol. To overcome competition with native pathways that share the same substrate, we engineered two bifunctional enzymes that redirect metabolic flux toward the synthetic pathway. Taken together, our work demonstrates a new approach to the engineering of novel synthetic pathways in the cell. The chemical and transportation industries currently use limited nonrenewable resources to produce raw materials that could instead be synthesized from renewable resources using metabolic engineering (18). Natural biological pathways have traditionally been the source of industrially important chemicals produced using fermentation (43). Some of the regulatory mechanisms that limit production in the native hosts are circumvented by reconstructing pathways in heterologous hosts (23). To synthesize chemicals not produced by natural pathways, enzymes are overexpressed in novel combinations in order to construct synthetic biological pathways (29,49).One of the rate-limiting steps in the assembly of a synthetic pathway is the identification of enzymes that can catalyze new reactions of interest. Reliance on known enzymatic reactions limits the number of synthetic pathways that could be assembled, and the task of engineering an enzyme to catalyze a novel reaction remains a challenge. Strategies that incorporate directed evolution or metagenomic libraries require product-specific high-throughput screens or selections in order to identify enzymes able to catalyze a desired reaction (17). Rational and in silico protein engineering strategies generate smaller libraries to alleviate the need for high-throughput technologies but require a priori knowledge about the protein being engineered (6). Therefore, the effort to discover an enzyme that can catalyze a desired reaction is often hindered by the lack of high-throughput screening or selection and insufficient knowledge about how to rationally engineer a protein.We provide a new framework to identify proteins able to catalyze a desired reaction on a new substrate based on models that describe pathway and enzyme evolution. The patchwork evolution model suggests that a new biological pathway is assembled by recruiting enzymes from existing pathways (16). The ability to assemble new pathways could be facilitated by the high degree of promiscuity demonstrated by enzymes. The promiscuity could be a source of divergence during enzyme evolution, since members of the same superfamily typically share the ...

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“…The sequence of the linker was selected according to a previous report. 16,17 To measure the biological activity of IFN(-MSA, gamma-activated sequence (GAS)-dependent signal transduction was used. Namely, four copies of GAS element (5 -AGTGATTTCTCGGAAAGAGAG-3 ) were inserted into multicloning site of pLuc-MCS (Stratagene, La Jolla, California).…”

Section: Plasmid Dnamentioning

confidence: 99%

Prolonged Circulation Half-life of Interferon γ Activity by Gene Delivery of Interferon γ–Serum Albumin Fusion Protein in Mice

Miyakawa¹,

Nishikawa²,

Takahashi³

et al. 2011

Journal of Pharmaceutical Sciences

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Control of protein functional dynamics by peptide linkers

Cited by 169 publications

References 107 publications

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Synthetic Pathway for Production of Five-Carbon Alcohols from Isopentenyl Diphosphate

Prolonged Circulation Half-life of Interferon γ Activity by Gene Delivery of Interferon γ–Serum Albumin Fusion Protein in Mice

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