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Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

ABSTRACT: Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D_9k. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear relationship between the linker length and the free energy of folding of the Ca²⁺-free protein. In contrast, the linker length has only a marginal effect on the Ca²⁺ affinity and cooperativity. The variant with a single-glycine extension displays slightly increased Ca²⁺ affinity, suggesting that the slightly extended linker allows optimized packing of the Ca²⁺-bound state. For the extreme case of disconnected subdomains, Ca²⁺ binding becomes coupled to folding and assembly. Still, a high affinity between the EF-hands causes the non-covalent pair to retain a relatively high apparent Ca²⁺ affinity. Our results imply that loop length variation could be an evolutionary option for modulating properties such as protein stability and turnover without compromising the energetics of the specific function of the protein.

SUBMITTER: Linse S

PROVIDER: S-EPMC7674454 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

Linse Sara S Thulin Eva E Nilsson Hanna H Stigler Johannes J

Scientific reports 20201118 1

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D<sub>9k</sub>. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear ...[more]

PMID: 33208843

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Project description:BACKGROUND: Protein structure prediction and computational protein design require efficient yet sufficiently accurate descriptions of aqueous solvent. We continue to evaluate the performance of the Coulomb/Accessible Surface Area (CASA) implicit solvent model, in combination with the Charmm19 molecular mechanics force field. We test a set of model parameters optimized earlier, and we also carry out a new optimization in this work, using as a target a set of experimental stability changes for single point mutations of various proteins and peptides. The optimization procedure is general, and could be used with other force fields. The computation of stability changes requires a model for the unfolded state of the protein. In our approach, this state is represented by tripeptide structures of the sequence Ala-X-Ala for each amino acid type X. We followed an iterative optimization scheme which, at each cycle, optimizes the solvation parameters and a set of tripeptide structures for the unfolded state. This protocol uses a set of 140 experimental stability mutations and a large set of tripeptide conformations to find the best tripeptide structures and solvation parameters. RESULTS: Using the optimized parameters, we obtain a mean unsigned error of 2.28 kcal/mol for the stability mutations. The performance of the CASA model is assessed by two further applications: (i) calculation of protein-ligand binding affinities and (ii) computational protein design. For these two applications, the previous parameters and the ones optimized here give a similar performance. For ligand binding, we obtain reasonable agreement with a set of 55 experimental mutation data, with a mean unsigned error of 1.76 kcal/mol with the new parameters and 1.47 kcal/mol with the earlier ones. We show that the optimized CASA model is not inferior to the Generalized Born/Surface Area (GB/SA) model for the prediction of these binding affinities. Likewise, the new parameters perform well for the design of 8 SH3 domain proteins where an average of 32.8% sequence identity relative to the native sequences was achieved. Further, it was shown that the computed sequences have the character of naturally-occuring homologues of the native sequences. CONCLUSION: Overall, the two CASA variants explored here perform very well for a wide variety of applications. Both variants provide an efficient solvent treatment for the computational engineering of ligands and proteins.

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Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

Publications

Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

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