Energy Landscape of Ubiquitin is Weakly Multidimensional

Although it is known that various intramolecular interactions determine protein mechanical stability, a detailed molecular-level understanding of the key regulators of protein mechanical stability is still lacking. Here, we present evidence for salt bridges in ubiquitin as important intramolecular interactions that can affect protein mechanical stability. Ubiquitin has two salt bridges: one relatively surface-exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52). Ubiquitin is a reversible post-translational modifier and is stable mechanically (F avg u = 185 pN). On breaking SB1, the mechanical stability of ubiquitin is slightly enhanced (F avg u = 193 pN). In contrast, the mechanical stability significantly decreased upon breaking SB2 (F avg u = 158 pN). These results suggest that SB1 are SB2 are regulators of the mechanical stability of ubiquitin. Interestingly, the mechanical stability decreased further (F avg u = 145 pN) for the double salt bridge (DB) null variant. Monte Carlo simulations elucidate that the main regulating factor is the spontaneous unfolding rate constant (k u 0 ), being the highest for the DB null variant followed by the SB2 null variant, and it remains unaltered for the SB1 null variant, while the native-to-transition-state distance (x u ) remains unchanged.Our study provides mechanistic understanding on how two native salt bridges can independently regulate the mechanical stability in a protein, which has implications in designing protein-based robust biomaterials in the future.

Section: ■ Results and Discussionmentioning

confidence: 99%

Native Salt Bridges Are a Key Regulator of Ubiquitin’s Mechanical Stability

Nandi

Ainavarapu

2022

“…Monte Carlo simulation based on the Bell-like model gives estimate of x u and k u 0 under the approximation that the free energy landscape is one-dimensional where pulling direction acts as the reaction coordinate. 25,26 It also assumes that the TS does not change its position along the reaction coordinate. 25 We have used the x u obtained in our previous work to finally compute the ΔG u # value from the reconstructed free energy profile of stretching and unfolding SUMO1.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Reconstruction of the Free Energy Profile for SUMO1 from Nonequilibrium Single-Molecule Pulling Experiments

Nandi

Ainavarapu

2022

Free energy profiles form the cornerstone in the study of protein folding and function. In this study, the free energy profile of SUMO1 protein is directly reconstructed using an extension of the Jarzynski equality from atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) experiments. SUMO1 is a ubiquitin-like posttranslational modifier protein having a β clamp motif in its structure, imparting it with mechanical stability. We use the Jarzynski equality to obtain the equilibrium free energy profile from repeated nonequilibrium single-molecule pulling experiments. Indeed, the free energy values determined by the Jarzynski equality are lesser than the normal work average at all extensions. The free energy profiles constructed for the two velocities (100 and 400 nm/s) overlap with each other. The unfolding free energy barrier is estimated to be ∼7.5 kcal/mol. We anticipate that the Jarzynski equality can be applied in a similar manner to other ubiquitin-like proteins to extract their differences in the free energy profile, and hence, the effect of sequence diversity of structurally homologous proteins on the free energy landscape can be studied.

“…These studies provide overwhelming evidence that the unfolding pathways at low and high forces are in many cases different (Figure 3), with a variety of unfolding pathways and intermediates that cannot be observed under high forces. [41][42][43]51,52 An interesting complement to the approaches discussed above can be provided by Brownian or Langevin dynamics simulations. These simulations can reproduce experimental-like traces of extension versus time.…”

Section: ■ the Low Force Regimementioning

confidence: 99%

Recent Advances and Emerging Challenges in the Molecular Modeling of Mechanobiological Processes

Stirnemann

2022

Many biological processes result from the effect of mechanical forces on macromolecular structures and on their interactions. In particular, the cell shape, motion, and differentiation directly depend on mechanical stimuli from the extracellular matrix or from neighboring cells. The development of experimental techniques that can measure and characterize the tiny forces acting at the cellular scale and down to the single-molecule, biomolecular level has enabled access to unprecedented details about the involved mechanisms. However, because the experimental observables often do not provide a direct atomistic picture of the corresponding phenomena, particle-based simulations performed at various scales are instrumental in complementing these experiments and in providing a molecular interpretation. Here, we will review the recent key achievements in the field, and we will highlight and discuss the many technical challenges these simulations are facing, as well as suggest future directions for improvement.