Rational Design of a Calcium-Binding Protein

Yang, Wei; Jones, Lisa M.; Isley, Leanne; Ye, Yiming; Lee, Hsiau‐Wei; Wilkins, Anna L.; Liu, Zhi-ren; Hellinga, Homme W.; Malchow, Russell; Ghazi, Mohammed; Yang, Jenny J.

doi:10.1021/ja034724x

Cited by 65 publications

(99 citation statements)

References 47 publications

(75 reference statements)

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“…33,44 The calcium dissociation constant for the Dezymer-redesigned protein CD2.Ca1 was reported to be 40 AE 10 lM, 33 which is similar to the calcium dissociation constant of 59 AE 2 lM for the bestredesigned OptGraft protein. Despite some position similarities between our designs and CD2.Ca1, the structural characteristics of these designs are distinctively The current implementation of OptGraft can be used to introduce only binding (not catalytic) pockets onto existing protein scaffolds as all the geometry optimization/energy minimization steps are performed only at the ground state.…”

Section: Summary and Discussionmentioning

confidence: 78%

“…This domain has been extensively used as a platform for different protein design endeavors, and it has been shown that it can accommodate different configurations of calcium-binding pockets. [32][33][34][35] Ye et al employed glycine linkers to fuse calcium-binding loop III from calmodulin at three different locations in the first domain of CD2 protein. 34 The results indicated that the redesigned protein gained calcium-binding centers while at the same time the overall conformation of the host protein was minimally disturbed.…”

Section: Optgraft Predictionsmentioning

confidence: 99%

“…Using the Dezymer algorithm, this domain has also been subjected to de novo design of calcium-binding pockets. 32,33,35 A calcium-binding pocket is usually located within a loop of 10-15 contiguous residues, with calcium coordinated by oxygen donors from the side-chain carboxylate and hydroxyl groups as well as peptide carbonyl oxygen atoms and water molecules. 36 In non-EF-hand-type calcium-binding proteins, which include proteins stabilized by calcium ions, the metal center is usually located on flexible X-shape surface loops between protein b-strands.…”

Section: Optgraft Predictionsmentioning

confidence: 99%

“…4). Interestingly, the location of these three designs shares four positions with the CD2.Ca1 redesigned protein constructed using Dezymer, 33 where a different set of mutations was introduced in a cluster of five amino acid positions (i.e., 21, 78, 80, 89, and 91) to construct a single calcium-binding center on the same protein scaffold.…”

Section: Optgraft Predictionsmentioning

confidence: 99%

“…[41][42][43] Direct binding of lanthanides can be monitored via FRET interactions between the metal and aromatic residues. Terbium fluorescence has been shown to serve as a suitable surrogate calcium-binding site probe in CD2 mutants Ca.CD2, 35 CD2.Ca1, 33 and DEEEE 32 designed for calcium binding. The protein scaffold used in this study (i.e., the first domain of CD2 protein) contains Trp32 and Tyr81 residues that are located in the proximity of the grafted binding pockets, and their fluorescence emissions can excite the bound terbium.…”

Section: Experimental Testingmentioning

confidence: 99%

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OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

2008

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One of the many challenging tasks of protein design is the introduction of a completely new function into an existing protein scaffold. In this study, we introduce a new computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. This is accomplished by introducing a two-level procedure where we first identify where are the most appropriate locations to graft the new binding pocket into the protein fold by minimizing the departure from a set of geometric restraints using mixed-integer linear optimization. On identifying the suitable locations that can accommodate the new binding pocket, CHARMM energy calculations are employed to identify what mutations in the neighboring residues, if any, are needed to ensure that the minimum energy conformation of the binding pocket conserves the desired geometry. This computational framework is benchmarked against the results available in the literature for engineering a copper binding site into thioredoxin protein. Subsequently, OptGraft is used to guide the transfer of a calcium-binding pocket from thermitase protein (PDB: 1thm) into the first domain of CD2 protein (PDB:1hng). Experimental characterization of three de novo redesigned proteins with grafted calcium-binding centers demonstrated that they all exhibit high affinities for terbium (K d $ 22, 38, and 55 lM) and can selectively bind calcium over magnesium.

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Section: Summary and Discussionmentioning

confidence: 78%

Section: Optgraft Predictionsmentioning

confidence: 99%

Section: Optgraft Predictionsmentioning

confidence: 99%

Section: Optgraft Predictionsmentioning

confidence: 99%

Section: Experimental Testingmentioning

confidence: 99%

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OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

2008

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Metalloprotein Design & Engineering

2005

Encyclopedia of Inorganic Chemistry

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This review covers recent advances in metalloprotein design, with focus on different approaches to the design. Impressive progress has been made in designing metal‐binding sites in peptides, de novo designed proteins, and native protein scaffolds. The approach can be rational or combinatorial. Under rational design, redesigning an existing metal‐binding site to a new site with dramatically different structure and function complements well the design of new metal‐binding sites by revealing the role of specific residues responsible for a particular structural or functional feature of the metal‐binding site of interest. To create a new metal‐binding site, several approaches have been used, including design based on structural homology, by inspection, using automated computer search algorithms, or combination of the above approaches. In addition, modular approach by transplanting a conserved structural unit from one protein into another has also been shown to be effective. Design through combinatorial and evolution methods has also been successful as it requires little prior knowledge of the protein structure. Finally, introducing unnatural amino acids or nonnative metal ions/prosthetic groups to expand the repertoires of metalloproteins have been demonstrated. Successful examples of each of the approaches are given; advantages and disadvantages of the approaches are discussed; the outlook for future research is also presented.

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Metalloprotein Design & Engineering

Lu¹

2005

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Rational Design of a Calcium-Binding Protein

Cited by 65 publications

References 47 publications

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

Metalloprotein Design & Engineering

Metalloprotein Design & Engineering

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