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Solvent molecules bridge the mechanical unfolding transition state of a protein.

ABSTRACT: We demonstrate a combination of single molecule force spectroscopy and solvent substitution that captures the presence of solvent molecules in the transition state structure. We measure the effect of solvent substitution on the rate of unfolding of the I27 titin module, placed under a constant stretching force. From the force dependency of the unfolding rate, we determine Deltax(u), the distance to the transition state. Unfolding the I27 protein in water gives a Deltax(u) = 2.5 A, a distance that compares well to the size of a water molecule. Although the height of the activation energy barrier to unfolding is greatly increased in both glycerol and deuterium oxide solutions, Deltax(u) depends on the size of the solvent molecules. Upon replacement of water by increasing amounts of the larger glycerol molecules, Deltax(u) increases rapidly and plateaus at its maximum value of 4.4 A. In contrast, replacement of water by the similarly sized deuterium oxide does not change the value of Deltax(u). From these results we estimate that six to eight water molecules form part of the unfolding transition state structure of the I27 protein, and that the presence of just one glycerol molecule in the transition state is enough to lengthen Deltax(u). Our results show that solvent composition is important for the mechanical function of proteins. Furthermore, given that solvent composition is actively regulated in vivo, it may represent an important modulatory pathway for the regulation of tissue elasticity and other important functions in cellular mechanics.

SUBMITTER: Dougan L

PROVIDER: S-EPMC2265161 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Solvent molecules bridge the mechanical unfolding transition state of a protein.

Dougan Lorna L Feng Gang G Lu Hui H Fernandez Julio M JM

Proceedings of the National Academy of Sciences of the United States of America 20080227 9

We demonstrate a combination of single molecule force spectroscopy and solvent substitution that captures the presence of solvent molecules in the transition state structure. We measure the effect of solvent substitution on the rate of unfolding of the I27 titin module, placed under a constant stretching force. From the force dependency of the unfolding rate, we determine Deltax(u), the distance to the transition state. Unfolding the I27 protein in water gives a Deltax(u) = 2.5 A, a distance tha ...[more]

PMID: 18305176

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Project description:Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The φ(M2+)(U)-value is defined as ΔΔG(‡-N)/ΔΔG(U-N), which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG(‡-N)) relative to its thermodynamic stability (ΔG(U-N)) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify φ(M2+)(U)-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1's mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.

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Solvent molecules bridge the mechanical unfolding transition state of a protein.

Publications

Solvent molecules bridge the mechanical unfolding transition state of a protein.

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