Single homopolypeptide chains collapse into mechanically rigid conformations

Abstract

Huntington's disease is linked to the insertion of glutamine (Q) in the protein huntingtin, resulting in polyglutamine (polyQ) expansions that self-associate to form aggregates. While polyQ aggregation has been the subject of intense study, a correspondingly thorough understanding of individual polyQ chains is lacking. Here we demonstrate a single molecule force-clamp technique that directly probes the mechanical properties of single polyQ chains. We have made polyQ constructs of varying lengths that span the length range of normal and diseased polyQ expansions. Each polyQ construct is flanked by the I27 titin module, providing a clear mechanical fingerprint of the molecule being pulled. Remarkably, under the application of force, no extension is observed for any of the polyQ constructs. This is in direct contrast with the random coil protein PEVK of titin, which readily extends under force. Our measurements suggest that polyQ chains form mechanically stable collapsed structures. We test this hypothesis by disrupting polyQ chains with insertions of proline residues and find that their mechanical extensibility is sensitive to the position of the proline interruption. These experiments demonstrate that polyQ chains collapse to form a heterogeneous ensemble of conformations that are mechanically resilient. We further use a heat-annealing molecular dynamics protocol to extensively search the conformation space and find that polyQ can exist in highly mechanically stable compact globular conformations. The mechanical rigidity of these collapsed structures may exceed the functional ability of eukaryotic proteasomes, resulting in the accumulation of undigested polyQ sequences in vivo.

Fig. 1.

A single molecule protocol to probe the mechanical properties of polypeptide chains (A) We construct the polyprotein (I27)₃ containing 3 identical repeats of the human cardiac titin Ig domain I27. Using force-clamp spectroscopy we apply a constant force of 180 pN to the construct resulting in a well defined series of step increases in length, marking the unfolding of each I27 protein in the chain. Complete extension of the construct is identified by the mechanical fingerprint of 3 step increases in length of 24 nm. An initial extension in length (L_Initial) is observed, corresponding to the elongation of the folded I27 proteins and the amino acid linkers in the (I27)₃ construct. A histogram of L_Initial is shown for all force-clamp trajectories (n = 93) containing 3 I27 unfolding events. A Gaussian fit to the histogram gives an average L_Initial = 13.30 ± 7.10 nm, in close agreement with the expected extension of 3 folded I27 proteins and the linkers in the construct (gray shaded area) (B) We construct the chimera (I27-PEVK-I27-PEVK-I27) containing the random coil protein PEVK in tandem with the I27 protein. We measure the mechanical properties by applying a constant force of 180 pN, resulting in a series of step increases in length. Each trajectory contains an initial extension L_Initial corresponding to the elongation of the folded I27 proteins, PEVK and linkers in the construct. A histogram of L_Initial is shown for force-clamp trajectories (n = 106) containing staircases of 3 I27 unfolding events with steps of 24 nm. A Gaussian fit to the histogram gives an average L_Initial = 40.65 ± 17.26 nm, in agreement with the expected extension of 3 folded I27 proteins, PEVK and the amino acid linkers in the construct (black shaded area).

Fig. 2.

Probing the mechanical properties of homopolypeptide chains. We construct chimeras containing the I27 protein and polyglutamine chains of different length, namely Q15 (A), Q25 (B) Q50 (C), and Q75 (D). Applying a constant force of 180 pN, results in a series of step increases in length of 24 nm. We identify the full mechanical extension of a complete construct by the presence of 3 I27 unfolding steps for the Q25, Q50, and Q75 constructs and 5 I27 unfolding steps for the Q15 construct (Table S1). We measure the initial extension L_Initial for each trajectory that satisfies these stringent criteria. A histogram of L_Initial is shown for each of the constructs; Q15 (n = 121), Q25 (n = 165), Q50 (n = 100), and Q75 (n = 149). A Gaussian fit to the histograms (solid line) gives an average L_Initial for each construct Q15 (L_Initial = 31.16 ± 8.21 nm), Q25 (L_Initial = 13.79 ± 9.63 nm), Q50 (L_Initial = 26.09 ± 7.93 nm), Q75 (L_Initial = 20.65 ± 6.98 nm), (QP)₂₄ (L_Initial = 42.3.1 ± 9.63 nm), and (Q11P)₄ (L_Initial = 70.00 ± 12.39 nm). For all polyglutamine chains, the measured L_Initial is significantly shorter than that expected for full extension of the construct (black shaded area). Instead L_Initial is in close agreement with the expected length extension of only the folded I27 proteins and linkers (gray shaded area).

Fig. 3.

Representative structures of polyglutamine chains display extreme mechanical stability (A) SMD simulations provide a detailed atomic picture of stretching individual polyQ chains. In the simulations, polyQ chains of length N_PP = 25 were first heated to a high temperature and then heat annealed to obtain collapsed structures. Each collapsed structure (N ≈10,000) was then pulled at a constant rate to trigger unraveling (see SI Text). Upon pulling, a number of configurations exhibit very high mechanical stability with rupture forces of more than 900 pN. Examples of these mechanically resilient structures are shown in (B and C) Upon close examination of the force induced unraveling of these mechanically resilient structures we find that a large number of hydrogen bonds must rupture simultaneously before unraveling occurs.

Fig. 4.

Probing the mechanical properties of heteropolypeptide chains. We construct chimeras containing the I27 protein and a heteropolypeptide chain containing glutamines interrupted by a proline residues, namely [I27-(QP)₂₄] and [I27-(Q11P)₄] (Table S1). Using force-clamp spectroscopy we apply a constant force of 180 pN along the end to end length of the constructs. We identify the full mechanical extension of a complete construct by the presence of 3 I27 unfolding steps for the (QP)₂₄ and (Q11P)₄ constructs (Table S1) where each I27 unfolding step is 24 nm in length. We measure the initial extension L_Initial for each trajectory that satisfies these stringent criteria. A histogram of L_Initial is shown for each of the constructs; (A) Q50 (n = 100) for comparison, (B) (QP)₂₄ (n = 108), and (C) (Q11P)₄ (n = 133). A Gaussian fit to the histograms (solid line) gives an average L_Initial for each construct (QP)₂₄ (L_Initial = 42.3.1 ± 9.63 nm) and (Q11P)₄ (L_Initial = 70.00 ± 12.39 nm). The measured L_Initial for the heteropolpeptide chain Q11P, is in agreement with the full extension of the folded I27 proteins, polypeptides, and linkers. Interestingly, L_Initial for the (QP)₂₄ construct lies between the gray and black shaded areas.

Fig. 5.

Homopolypeptide chains display extreme mechanical resilience under the application of a high force (A) To further examine the mechanical properties of the polyglutamine chain we extended the force regime with which we probed the system. We used a force-ramp protocol to apply a linearly increasing force to the (I27-Q50) construct. In this force protocol we ramp the force from 0 pN to 1000 pN over a time period of 15 seconds. Extension of the (I27-Q50) construct was identified by the presence of 3 I27-unfolding steps in the length trajectory. Unfolding of the I27 protein occurred between a force of 150 pN and 200 pN, in accord with the known mechanical properties of this protein. Using this protocol, extension of the polyglutamine chain would be identified by a marked increase in length of approximately 40 nm (N_PP x L_AA) (B) We measured the final extension of the (I27-Q50) construct for each trajectory where the full I27-unfolding fingerprint could be identified. The average final extension approximately116 nm was much lower than that expected for the complete extension of 3 unfolded I27 proteins (89 × L_AA), 100 glutamines (N_PP× L_AA) and the linkers (N_Link× L_AA) (solid black line). Instead, the final extension was in close agreement with the extension of only the unfolded I27 proteins and linkers. Thus, no evidence for the extension of the polyglutamine chain was observed, even at force as high as 800 pN suggesting that the polyglutamine chains possess extreme mechanical stability.