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

Sara Linse,Johannes Stigler,Eva Thulin,Hanna Nilsson

doi:10.1038/s41598-020-76598-x

Sara Linse, Johannes Stigler + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-76598-x

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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 D9k. 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.

Highlights

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain
We monitored the far-UV circular dichroism (CD) signal at 222 nm, which is mainly used as a reporter for helicity in folded polypeptides, as it changes upon increasing the concentration of urea in the buffer from 0 to 9.75 M (Fig. 2a)
The m-values appear to be independent of linker length (Pearson’s r = 0.22 ± 0.31, slope = 0.002 ± 0.009, Fig. 2b) and average at 4.28 ± 0.03 kJ/(mol M)

Summary

Introduction

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. The native structure can still remain unaffected when the chain topology is altered by circular permutation, i.e. linking of N- and C-termini while cutting another loop, albeit at the expense of an altered folding p athway[12] These examples illustrate the importance of non-covalent interactions in governing the native fold of a protein. The first classical example of this property of fragment complementation is ribonuclease which was reconstituted 1958 from two separate polypeptide fragments with retained fold and function[13] This exercise has been followed by many reports of proteins that are stable enough that their structure and function can be reconstituted through the non-covalent assembly of fragments comprising two or more s ubdomains[14,15,16,17,18]. This phenomenon is not limited to dimers; higher order oligomers may form through domains swapping and the phenomenon may even lead to the formation of gels or extended aggregates via runaway-domains s wapping[21,22]

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