How Do Chemical Denaturants Affect the Mechanical Folding and Unfolding of Proteins?

Cao, Yi; Li, Hongbin

doi:10.1016/j.jmb.2007.10.024

Cited by 61 publications

(57 citation statements)

References 52 publications

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“…We propose a mechanism where urea has a direct action on the mechanical stability of PKD domains by weakening these force-bearing hydrogen bonds. A similar mechanism was proposed for the effect of guanidinium chloride on the mechanical stability of the B1 immunoglobulin-binding domain of protein G (35).…”

Section: Discussionsupporting

confidence: 53%

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Naturally Occurring Osmolytes Modulate the Nanomechanical Properties of Polycystic Kidney Disease Domains

Oberhauser

2010

Journal of Biological Chemistry

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Polycystin-1 (PC1) is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. However, it is known that the native conformation of proteins can be stabilized by protecting osmolytes that are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here, we used single-molecule force spectroscopy to study directly the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and trimethylamine N-oxide can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated polycystin-2 channel or other intracellular events mediated by PC1. Polycystin-1 (PC1)2 is a large transmembrane protein, which, when mutated, cause autosomal dominant polycystic kidney disease (PKD), one of the most common lifethreatening genetic diseases, which is a leading cause of kidney failure (1). The available evidence suggests that PC1 acts as a mechanosensor, receiving signals from the primary (luminal) cilia, neighboring cells, and extracellular matrix and transduces them into cellular responses that regulate proliferation, adhesion, and differentiation that are essential for the control of renal tubules and kidney morphogenesis (1-4).PC1 is expressed along the renal tubule, where it is exposed to a wide range of concentrations of urea, from 5 mM in the proximal tubule to up to ϳ1 M in the collecting duct (5-7). Urea is known as a common denaturant that affects protein function by perturbing their structure (8). Several osmolytes have been found in the mammalian kidney that are known to counteract the effects of urea on proteins (9 -15). Among epithelial cells of the kidney medulla, the principal organic osmolytes include the polyols sorbitol and inositol, the methylamines betaine and glycerophosphocholine, and the amino acid taurine. Protecting osmolytes are found in all taxa (11,14). For example, cartilaginous fish concentrate trimethylamine N-oxide (TMAO), to offset the damaging effects of urea on protein function, and it is one of the best studied protecting osmolytes (e.g. 16 -20).The mecha...

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Section: Discussionsupporting

confidence: 53%

“…This experimental approach more closely mimics the conditions found in vivo. In addition, single-molecule AFM experiments have provided novel and valuable insights into how osmolytes affect protein unfolding/folding dynamics (34,35).…”

Section: Polycystin-1 (Pc1)mentioning

confidence: 99%

Naturally Occurring Osmolytes Modulate the Nanomechanical Properties of Polycystic Kidney Disease Domains

Oberhauser

2010

Journal of Biological Chemistry

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“…Denaturing osmolytes such as guanidine hydrochloride (GdnHCl) and urea shift the equilibrium to the unfolded state of the protein. SMFS studies have been performed in solvent environments containing a number of different osmolytes [214][215][216]. For example, glycerol has been shown to increase the mechanical stability of the protein GB1 [216], while denaturing osmolytes such as GdnHCl and urea have been found to decrease the mechanical stability of proteins [215,217].…”

Section: Role Of Solvent Interactions On Protein Mechanical Stabilitymentioning

confidence: 99%

“…SMFS studies have been performed in solvent environments containing a number of different osmolytes [214][215][216]. For example, glycerol has been shown to increase the mechanical stability of the protein GB1 [216], while denaturing osmolytes such as GdnHCl and urea have been found to decrease the mechanical stability of proteins [215,217]. The mechanical properties of a protein can be tuned by a change in pH or ionic strength of a buffer [81,81,218].…”

Section: Role Of Solvent Interactions On Protein Mechanical Stabilitymentioning

confidence: 99%

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

2016

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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.

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“…various proteins (28)(29)(30)(31)(32), and hydrophobic homopolymers such as poly (methyl methacrylate) (33) and polystyrene (34)(35)(36) has proven to be of substantial value in answering questions on the nature of hydrophobic interactions under different conditions.…”

Section: Significancementioning

confidence: 99%

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory

Mondal

Halverson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

119

153

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It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford-Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard-Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system. single-molecule force spectroscopy | free energy | preferential binding | TMAO | urea

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How Do Chemical Denaturants Affect the Mechanical Folding and Unfolding of Proteins?

Cited by 61 publications

References 52 publications

Naturally Occurring Osmolytes Modulate the Nanomechanical Properties of Polycystic Kidney Disease Domains

Naturally Occurring Osmolytes Modulate the Nanomechanical Properties of Polycystic Kidney Disease Domains

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

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory

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