Towards design principles for determining the mechanical stability of proteins

Hoffmann, T. A.; Tych, Katarzyna M.; Hughes, Megan L.; Brockwell, David J.; Dougan, Lorna

doi:10.1039/c3cp52142g

Cited by 60 publications

(95 citation statements)

References 102 publications

(200 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This study demonstrates that similarities in the structural topologies of proteins results in similarities in the mechanical unfolding energy landscapes. The relationship between the x U and F has been shown to follow a power law suggesting proteins with a lower unfolding force have a higher x U (forces determined at 600 nm/s) [229]. These studies also reveal gaps in the explored space of mechanical properties of studied proteins (Figure 26), which will be helpful for the selection of proteins for future force spectroscopy studies.…”

Section: Mechanical Phi-value Analysis Probes Mechanical Unfolding Trmentioning

confidence: 92%

“…This offers an attractive, high throughput tool for identifying target proteins for desired applications where knowledge of the mechanical properties are required in a timely and accurate manner without the need for time-intensive experiments. This reveals features such as a correlation between the type of mechanical clamp in a protein and its mechanical properties [229]. For example, the ∆x U and the unfolding force ( Figure 26).…”

Section: Mechanical Phi-value Analysis Probes Mechanical Unfolding Trmentioning

confidence: 99%

See 1 more Smart Citation

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

2016

Self Cite

View full text Add to dashboard Cite

One of the most exciting developments in the field of biological physics in recent years is the ability to manipulate single molecules and probe their properties and function. Since its emergence over two decades ago, single molecule force spectroscopy has become a powerful tool to explore the response of biological molecules, including proteins, DNA, RNA and their complexes, to the application of an applied force. The force versus extension response of molecules can provide valuable insight into its mechanical stability, as well as details of the underlying energy landscape. In this review we will introduce the technique of single molecule force spectroscopy using the atomic force microscope (AFM), with particular focus on its application to study proteins. We will review the models which have been developed and employed to extract information from single molecule force spectroscopy experiments. Finally, we will end with a discussion of future directions in this field.

show abstract

Section: Mechanical Phi-value Analysis Probes Mechanical Unfolding Trmentioning

confidence: 92%

Section: Mechanical Phi-value Analysis Probes Mechanical Unfolding Trmentioning

confidence: 99%

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…The mechanical strength of these homologous proteins was then compared as the relationship between thermodynamic stability and mechanical properties of a protein remains unclear. 40,48 SMFS experiments were completed using the chimeric polyprotein (I27-BsCSP) 3 -I27 (Fig. 2(c)) and compared to previously obtained mechanical unfolding data for TmCSP in an analogous scaffold ((I27-TmCSP) 3 -I27).…”

Section: Resultsmentioning

confidence: 99%

Tuning protein mechanics through an ionic cluster graft from an extremophilic protein

et al. 2016

Self Cite

View full text Add to dashboard Cite

Proteins from extremophilic organisms provide excellent model systems to determine the role of non-covalent interactions in defining protein stability and dynamics as well as being attractive targets for the development of robust biomaterials. Hyperthermophilic proteins have a prevalence of salt bridges, relative to their mesophilic homologues, which are thought to be important for enhanced thermal stability. However, the impact of salt bridges on the mechanical properties of proteins is far from understood. Here, a combination of protein engineering, biophysical characterisation, single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations directly investigates the role of salt bridges in the mechanical stability of two cold shock proteins; BsCSP from the mesophilic organism Bacillus subtilis and TmCSP from the hyperthermophilic organism Thermotoga maritima. Single molecule force spectroscopy shows that at ambient temperatures TmCSP is mechanically stronger yet, counter-intuitively, its native state can withstand greater deformation before unfolding (i.e. it is mechanically soft) compared with BsCSP. MD simulations were used to identify the location and quantify the population of salt bridges, and reveal that TmCSP contains a larger number of highly occupied salt bridges than BsCSP. To test the hypothesis that salt-bridges endow these mechanical properties on the hyperthermophilic CSP, a charged triple mutant (CTM) variant of BsCSP was generated by grafting an ionic cluster from TmCSP into the BsCSP scaffold. As expected CTM is thermodynamically more stable and mechanically softer than BsCSP. We show that a grafted ionic cluster can increase the mechanical softness of a protein and speculate that it could provide a mechanical recovery mechanism and that it may be a design feature applicable to other proteins.

show abstract

“…Interestingly, this is a typical feature of mechanically stable proteins. 62 These forcebearing strands are connected by hydrogen bonds; our simulations show approximately six hydrogen bonds on average between -strands 1-4 and seven between -strands 4-5 (Fig. 2b), and they may represent the 'mechanical clamp' of the TmCSP protein.…”

Section: Discussionmentioning

confidence: 99%

Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein

et al. 2016

Self Cite

View full text Add to dashboard Cite

Article:Tych, KM, Batchelor, M orcid.org/0000-0001-6338-5698, Hoffmann, T et al. KEYWORDS AFM, extremophile, mechanical stability, cold shock proteins ABSTRACT Proteins from organisms which have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on hyperthermophilic protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal -strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (∆GD-N) of the mutated proteins and show there is a more significant reduction for TmCSP L40A (∆∆G = 63%) than TmCSP V62A (∆∆G = 47%) as might be expected, based on the relative reduction in the size of the side chain. By contrast SMFS measures the mechanical stability (∆G*) and shows a greater reduction for TmCSP V62A (∆∆G* = 8.4%) than TmCSP L40A (∆∆G* = 2.5%). While the impact on mechanical stability is subtle, the results demonstrate the power of tuning non-covalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provides the opportunity to design proteins with optimized thermodynamic and mechanical properties. INTRODUCTIONProteins from organisms adapted to high-temperatures have evolved to retain their native, folded structure and function in the challenging environments in which the organisms grow.

show abstract

Towards design principles for determining the mechanical stability of proteins

Cited by 60 publications

References 102 publications

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

Tuning protein mechanics through an ionic cluster graft from an extremophilic protein

Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein

Contact Info

Product

Resources

About