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

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 (Favgu = 185 pN). On breaking SB1, the mechanical stability of ubiquitin is slightly enhanced (Favgu = 193 pN). In contrast, the mechanical stability significantly decreased upon breaking SB2 (Favgu = 158 pN). These results suggest that SB1 are SB2 are regulators of the mechanical stability of ubiquitin. Interestingly, the mechanical stability decreased further (Favgu = 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 (ku0), 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 (xu) 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.

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

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 ß 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.

Rational Design of Protein-Specific Folding Modifiers.

Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

Applications of atomic force microscopy in modern biology.

Single-molecule force spectroscopy (SMFS) is an emerging tool to investigate mechanical properties of biomolecules and their responses to mechanical forces, and one of the most-used techniques for mechanical manipulation is the atomic force microscope (AFM). AFM was invented as an imaging tool which can be used to image biomolecules in sub-molecular resolution in physiological conditions. It can also be used as a molecular force probe for applying mechanical forces on biomolecules. In this brief review, we will provide exciting examples from recent literature which show how the advances in AFM have enabled us to gain deep insights into mechanical properties and mechanobiology of biomolecules. AFM has been applied to study mechanical properties of cells, tissues, microorganisms, viruses as well as biological macromolecules such as proteins. It has found applications in biomedical fields like cancer biology, where it has been used both in the diagnostic phases as well as drug discovery. AFM has been able to answer questions pertaining to mechanosensing by neurons, and mechanical changes in viruses during infection by the viral particles as well as the fundamental processes such as cell division. Fundamental questions related to protein folding have also been answered by SMFS like determination of energy landscape properties of variety of proteins and their correlation with their biological functions. A multipronged approach is needed to diversify the research, as a combination with optical spectroscopy and computer-based steered molecular dynamic simulations along with SMFS can help us gain further insights into the field of biophysics and modern biology.

The unfolding transition state of ubiquitin with charged residues has higher energy than that with hydrophobic residues.

The native-state structure and folding pathways of a protein are encoded in its amino acid sequence. Ubiquitin, a post-translational modifier, primarily noted for its role in intracellular protein degradation, has two salt bridges: one relatively exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52). Here, we study the role of hydrophobic interactions and sequence specificity in protein folding, by mutating the salt-bridge residues in ubiquitin with hydrophobic residues. Equilibrium chemical denaturation using GdnHCl shows that the SB1 null variant is thermodynamically stabilised whereas the SB2 null variant is destabilised only slightly. The thermodynamic stability of the double salt-bridge (DB) null variant is an additive effect of the individual salt bridges. Kinetic experiments show that all the salt-bridge null variants fold through a more stable intermediate with relatively faster folding rates than the wild-type. The SB2 null variant has a highly stabilised unfolding transition state (TS) and a slightly destabilised native state, leading to its kinetic instability, whereas the kinetic stability of the SB1 null variant is not compromised as its TS and native state are stabilized to a similar extent. The TS stabilisation is also additive for the DB null variant, which has the most stabilised TS and high kinetic instability. Our results underscore the importance of kinetic stability in optimising the protein energy landscape. Our study establishes the fact that the TSs can be stabilized by hydrophobic residues in the place of buried charged residues. It further highlights the role of charged residues in the protein interior in dictating the folding pathway.

Experimental comparison of energy landscape features of ubiquitin family proteins.

Small ubiquitin-related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence-structure-stability-function relationship. Here, we used chemical denaturation in comparing the folding-unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W-pseudo wild-type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant-dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two-state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow-down their folding rates. The denaturant-dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent-accessible surface area, as quantified by the Tanford ß u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding-unfolding rates within a structurally homologous protein family.