Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein

Abstract

Although multidomain proteins predominate the proteome of all organisms and are expected to display complex folding behaviors and significantly greater structural dynamics as compared with single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands are poorly understood due to a lack of experimental techniques. The multidomain calcium-binding ␤␥-crystallin proteins are particularly important because their deterioration and misfolding/aggregation are associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding ␤␥-crystallin protein, Protein S, and elaborate on its interactions with calcium. We ask whether domain interactions and calcium binding affect Protein S folding and potential structural heterogeneity. Our results from single-molecule force spectroscopy show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca 2؉ , which is mechanically stable in contrast to other proteins that were observed to sample a molten globule under similar conditions. Mutagenesis experiments and computer simulations reveal that the alternative conformation of the N-terminal domain is caused by structural instability produced by the high charge density of a calcium binding site. We find that this alternative conformation in the N-terminal domain is diminished in the presence of calcium and can also be partially eliminated with a hitherto unrecognized compensatory mechanism that uses the interaction of the C-terminal domain to neutralize the electronegative site. We find that up to 1% of all identified multidomain calcium-binding proteins contain a similarly highly charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand.Multidomain proteins are highly prevalent in the proteomes of all organisms (1); however, there is still little known about their folding pathways and interaction with ligands (2), as compared with small, single-domain proteins. This is because large, multidomain proteins may exhibit significant structural dynamics and transient interactions between the domains that are rather difficult to capture and analyze. An important example of this class of proteins are the calcium-binding proteins that are effectors for stimulating cellular signals (3, 4) and buffering calcium levels (5). Currently, of particular interest are the calcium-binding ␤␥-crystallin proteins that are implicated in several human diseases (6 -8). The complexity of these proteins may best be understood through single-molecule methods that are able to deconvolve their conformational heterogeneity. Although there have been exciting recent single-molecule studies of the multidomain calmodulin, including the impact of domains' interactions on calcium binding (9), and studies on mechanical unfolding of ␤␥-crystallins that captured their interesting domain swapping behavior (10), ther...

Manipulating polydispersity of lens β-crystallins using divalent cations demonstrates evidence of calcium regulation

“…We are not the only ones to observe specific divalent cation-binding interactions within the β-subtype. In a study using a β-crystallin homolog, protein S sequenced from Myxococcus xanthus , Scholl et al found that the presence of calcium stabilizes a single conformation of the N-terminal domain ( 77 ). Without calcium, the N-terminal domain can adopt two alternative conformations.…”

“…This function appears to be evolutionarily conserved in the double Greek-key-folded structure ( 14 , 74 , 84 , 87 ). The major effect of cation binding in these other proteins is the increased thermodynamic, kinetic, and mechanical stability ( 72 , 74 , 75 , 77 , 85 , 86 ). The ability of lens β-crystallins to bind calcium has been speculated as a protective and homeostatic function.…”

Single-Chain Inherent Elasticity of Macromolecules: From Concept to Applications

“…In the middle of the 20th century, Flory and other pioneers put forward several statistical mechanical models to semiquantitatively describe the single-chain elasticities of polymers in theory. , With the advent of single-molecule atomic force microscopy (AFM), the elastic behaviors of individual macromolecules have been extensively researched experimentally since the 1990s. − Although great progress has been made toward understanding the single-chain properties of macromolecules, many challenges still remain in this field. − With a backbone and side chains, a polymer chain is more complex than a small molecule. Moreover, a single polymer chain is usually placed in a complicated environment such as solvent, cosolute, and solid surface, which significantly affects the behaviors of the chain.…”

Unraveling the Mechanical Unfolding Pathways of a Multidomain Protein: Phosphoglycerate Kinase

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“…One of the simplest SMFS methods is atomic force microscopy (AFM)-based SMFS, also known as atomic force spectroscopy (AFS). This technique has already proven very useful in examining proteins' unfolding and refolding pathways and capturing important details such as transient unfolding intermediates (61) while minimizing protein aggregation (31), making it an ideal tool for studying the mechanical stability and unfolding-refolding behavior of multidomain proteins. Recent improvements by the Perkins group in AFS force resolution and stability and the development of new powerful assays for specific attachment of molecules have significantly increased the AFS success rate, making AFS also ideally suited for studying mechanically weak and fast folding proteins (62,63).…”