Circular permutation of the starch-binding domain: inversion of ligand selectivity with increased affinity

Stephen, Preyesh; Tseng, Kai-Li; Liu, You‐Nian; Lyu, Ping‐Chiang

doi:10.1039/c2cc17376j

Cited by 11 publications

(13 citation statements)

References 28 publications

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“…The molecular mass of the mutant protein was determined by matrix-assisted laser desorption/ionization mass spectroscopy, which was in agreement with the theoretical molecular mass. The secondary structure content of Y52A verified by CD spectroscopy also was in agreement to the observed secondary structure of CP90 [33]. Using ITC, the binding curve for Y52A and amylose EX-I was best fit with a one-site binding equation (N = 0.5) and a K d value of 294 µM, indicating that the binding site in CP90 that has the greater affinity for amylose EX-I was absent in Y52A (Figure 6 and Table 4).…”

Section: Resultssupporting

confidence: 73%

“…The nucleotide sequence of Ro CBM21 [10] was used for getting the clone of CP90 in pET28a at Nco I and Xho I [33]. The mutant Y52A was obtained by point mutation using complementary primers containing the desired mutations (Forward: 5- GTCAAGAACATTGCTGCCTCCAAGAAAGTTACT-3 and reverse: 5- AGTAACTTTCTTGGAGGCAGCAATGTTCTTGAC-3) and Pfu ultra DNA polymerase (Agilent).…”

Section: Methodsmentioning

confidence: 99%

“…An efficient and selective attack by the GA on the crude mixture of starch molecule is a rate-limiting step in the aforementioned applications. Protein-engineering of SBDs [33] and amylase [34], [35] to obtain candidates that can efficiently and selectively attack specific form of carbohydrates may find useful applications in starch processing industries.…”

Section: Introductionmentioning

confidence: 99%

“…This mutant, whose N- terminus starts with G90 of Ro CBM21, was named CP90 [33]. Unlike most other constructed circularly permutated proteins [36]–[39], CP90 had significantly enhanced binding affinity and improved selectivity toward long-chain carbohydrates (i.e., its affinity for β-cyclodextrin is less than that for amylose EX-I, which is less than that for soluble and insoluble starches) [33]. For the study reported herein, examination of the CP90 crystal structure, its biophysical properties, and its binding affinity for amylose EX-I has suggested a possible, alternative binding path that causes its altered binding behavior.…”

Section: Introductionmentioning

confidence: 99%

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Crystal Structure of Circular Permuted RoCBM21 (CP90): Dimerisation and Proximity of Binding Sites

2012

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Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.

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Section: Resultssupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Crystal Structure of Circular Permuted RoCBM21 (CP90): Dimerisation and Proximity of Binding Sites

2012

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“…This program is designed to rank possible CP sites in a given protein based on structural coordinates and output scores for each residue, thus predicting which CPs will fold properly. Using this program, we have successfully predicted the folding of both naturally occurring and artificially designed CPs [14], [16], demonstrating the superior abilities of the CPred program.…”

Section: Introductionmentioning

confidence: 97%

Circular Permutation Prediction Reveals a Viable Backbone Disconnection for Split Proteins: An Approach in Identifying a New Functional Split Intein

Lee

et al. 2012

PLoS ONE

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Split-protein systems have emerged as a powerful tool for detecting biomolecular interactions and reporting biological reactions. However, reliable methods for identifying viable split sites are still unavailable. In this study, we demonstrated the feasibility that valid circular permutation (CP) sites in proteins have the potential to act as split sites and that CP prediction can be used to search for internal permissive sites for creating new split proteins. Using a protein ligase, intein, as a model, CP predictor facilitated the creation of circular permutants in which backbone opening imposes the least detrimental effects on intein folding. We screened a series of predicted intein CPs and identified stable and native-fold CPs. When the valid CP sites were introduced as split sites, there was a reduction in folding enthalpy caused by the new backbone opening; however, the coincident loss in entropy was sufficient to be compensated, yielding a favorable free energy for self-association. Since split intein is exploited in protein semi-synthesis, we tested the related protein trans-splicing (PTS) activities of the corresponding split inteins. Notably, a novel functional split intein composed of the N-terminal 36 residues combined with the remaining C-terminal fragment was identified. Its PTS activity was shown to be better than current reported two-piece intein with a short N-terminal segment. Thus, the incorporation of in silico CP prediction facilitated the design of split intein as well as circular permutants.

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Advances in molecular engineering of carbohydrate-binding modules

et al. 2017

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Carbohydrate-binding modules (CBMs) are non-catalytic domains that are generally appended to carbohydrate-active enzymes. CBMs have a broadly conserved structure that allows recognition of a notable variety of carbohydrates, in both their soluble and insoluble forms, as well as in their alpha and beta conformations and with different types of bonds or substitutions. This versatility suggests a high functional plasticity that is not yet clearly understood, in spite of the important number of studies relating protein structure and function. Several studies have explored the flexibility of these systems by changing or improving their specificity toward substrates of interest. In this review, we examine the molecular strategies used to identify CBMs with novel or improved characteristics. The impact of the spatial arrangement of the functional amino acids of CBMs is discussed in terms of unexpected new functions that are not related to the original biological roles of the enzymes. Proteins 2017; 85:1602-1617. © 2017 Wiley Periodicals, Inc.

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Circular permutation of the starch-binding domain: inversion of ligand selectivity with increased affinity

Cited by 11 publications

References 28 publications

Crystal Structure of Circular Permuted RoCBM21 (CP90): Dimerisation and Proximity of Binding Sites

Crystal Structure of Circular Permuted RoCBM21 (CP90): Dimerisation and Proximity of Binding Sites

Circular Permutation Prediction Reveals a Viable Backbone Disconnection for Split Proteins: An Approach in Identifying a New Functional Split Intein

Advances in molecular engineering of carbohydrate-binding modules

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