The effect of simple shear flow on the helix–coil transition of poly‐L‐lysine

Lee, A. T.; McHugh, A. J.

doi:10.1002/(sici)1097-0282(199911)50:6<589::aid-bip3>3.3.co;2-v

Cited by 6 publications

(10 citation statements)

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“…Force microscopy imaging of a very large plasma glycoprotein (vWF, a multimeric enzyme with molecular weight as large as 2 3 10 7 ) adhering to a surface seemed to reveal a shear-induced conformational transition (although not necessarily unfolding) occurring at a shear stress s ¼ h _ g g % 3.5 Pa (16), or _ g g ¼ s=h % 3:5310 3 s ÿ1 . In one of the most careful studies in this area, Lee and McHugh investigated the effect of simple shear on the helix-coil transition of poly-L-lysine (17). For solvent conditions that placed the sample very near the midpoint of its equilibrium helix-coil transition, they observed loss of helicity occurring at a critical shear rate _ g g; 300-400 s ÿ1 in a Couette flow cell.…”

Section: Introductionmentioning

confidence: 99%

Do Protein Molecules Unfold in a Simple Shear Flow?

Jaspe

Hagen

2006

Biophysical Journal

178

168

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Protein molecules typically unfold (denature) when subjected to extremes of heat, cold, pH, solvent composition, or mechanical stress. One might expect that shearing forces induced by a nonuniform fluid flow would also destabilize proteins, as when a protein solution flows rapidly through a narrow channel. However, although the protein literature contains many references to shear denaturation, we find little quantitative evidence for the phenomenon. We have investigated whether a high shear can destabilize a small globular protein to any measurable extent. We study a protein (horse cytochrome c, 104 amino acids) whose fluorescence increases sharply upon unfolding. By forcing the sample through a silica capillary (inner diameter 150-180 microm) at speeds approaching 10 m/s, we subject the protein to shear rates dv(z)/dr as large as approximately 2 x 10(5) s(-1) while illuminating it with an ultraviolet laser. We can readily detect fluorescence changes of <1%, corresponding to shifts of < approximately 0.01 kJ/mol in the stability of the folded state. We find no evidence that even our highest shear rates significantly destabilize the folded protein. A simple model suggests that extraordinary shear rates, approximately 10(7) s(-1), would be required to denature typical small, globular proteins in water.

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Section: Introductionmentioning

confidence: 99%

Do Protein Molecules Unfold in a Simple Shear Flow?

Jaspe

Hagen

2006

Biophysical Journal

178

168

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“…PLL can exist among three types of conformations, i.e., α-helix, β-sheet, and random coil, depending on the external environments (e.g., solvents and temperatures). − The conformational transition of the free-chain PLL in solutions has been studied quite extensively in the past. It has shown that conformational transitions can be induced by various external stimuli, including pH, , surfactants, − anions, − polyanions, − solvents, , heat, , mechanical shear force, etc. In addition, PLL has provided functions in gene delivery vectors, , biomimetic mineralization, , cell attachment, and biosensors and biosensor arrays .…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Conformational Transition of Surface-Tethered Polypeptide: Poly(l-lysine)

2003

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Poly(l-lysine) (PLL) was end-grafted onto silicon oxide surfaces to study its conformational transition among α-helix, β-sheet, and random coil at the solid−water interface. Surface-grafted PLL films were prepared from surface-grafted poly(N ε -carbobenzyloxy-l-lysine) (PCBL) thin films, which were fabricated by the surface-initiated vapor-deposition polymerization (SI-VDP) of N-carboxyanhydride (NCA) of N ε -carbobenzyloxy-l-lysine. The reaction parameters during the SI-VDP process, including NCA concentration, substrate temperature, and reaction time, were optimized in order to synthesize homogeneous PCBL films with well-controlled thickness. Under the optimal conditions (substrate temperature at 105 °C, NCA evaporating temperature at 100 °C, and 8 mg of NCA at 0.1 Pa), α-helical PCBL films with thickness ranging from 4 to 120 nm can be synthesized by controlling the reaction time from 5 to 120 min. The conversion from PCBL to PLL was accomplished by removing N ε -carbobenzyloxy groups in the hydrogen bromide/benzene solution with the aid of ultrasonication for 40 min. The surface-grafted PLL films with an estimated DP of 750 exhibited versatile conformations among α-helix, β-sheet, and random coil depending on the external environments. The conformational transition from one state to another was successfully induced by external stimuli, such as pH (H+/OH-), surfactant (sodium dodecyl sulfate (SDS)), and anion (ClO4 -). In the process of the conformational transition, macroscopic properties such as thickness, refractive index, and wettability changed correspondingly. For example, a surface-grafted random-coiled PLL had a film thickness of 215 nm and a refractive index of 1.37 at pH 7, whereas it changed to a β-sheet conformation when binding with SDS, with the film thickness shrinking to 145 nm and the refractive index increasing to 1.45. Compared to the free PLL chains, the stability of the helical state increased when the PLL chains were densely immobilized on the surfaces. The transitional behaviors were characterized by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and ellipsometry.

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“…A disadvantage of the benchtop shear cell was that we could not use NMR to monitor the reaction in real time; however, we were able to obtain a spectrum during an experiment by transferring the sample to a conventional NMR tube. Although a number of studies have highlighted the reversibility of shear-induced conformational changes upon cessation of shear (21,25), we expect that in this case, any significant changes that occurred during shearing of the sample in the benchtop cell would have left their mark by differences in the degree to which the polysaccharide chains would have been demethylesterified.…”

Section: Shearing Protocolsmentioning

confidence: 99%

“…In particular, it is worth noting that interfacial forces are much closer in magnitude to those found to be active in conformational manipulation in atomic force microscopy and optical tweezers experiments, compared with those that might be expected from shear flows (16). Nevertheless, recent well-controlled studies using modern optical and spectroscopic methods have also reported conformational changes in monomeric proteins (21)(22)(23)(24)(25)(26). A resolution of this issue is needed and could have far-reaching implications, such as for understanding the genesis of amyloidic diseases (24).…”

Section: Biopolymers Under Shearmentioning

confidence: 99%

Rheo-NMR Studies of an Enzymatic Reaction: Evidence of a Shear-Stable Macromolecular System

Edwards

Kakubayashi

Dykstra

et al. 2010

Biophysical Journal

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Understanding the effects of shear forces on biopolymers is key to understanding how biological systems function. Although currently there is good agreement between theoretical predictions and experimental measurements of the behavior of DNA and large multimeric proteins under shear flow, applying the same arguments to globular proteins leads to the prediction that they should only exhibit shear-induced conformational changes at extremely large shear rates. Nevertheless, contradictory experimental evidence continues to appear, and the effect of shear on these biopolymers remains contentious. Here, a custom-built rheo-NMR cell was used to investigate whether shear flow modifies enzyme action compared with that observed quiescently. Specifically, (1)H NMR was used to follow the kinetics of the liberation of methanol from the methylesterified polysaccharide pectin by pectinmethylesterase enzymes. Two different demethylesterifying enzymes, known to have different action patterns, were used. In all experiments performed, Couette flows with shear rates of up to 1570 s(-1) did not generate detectable differences in the rate of methanol liberation compared to unsheared samples. This study provides evidence for a shear-stable macromolecular system consisting of a largely beta-sheet protein and a polysaccharide, in line with current theoretical predictions, but in contrast to some other experimental work on other proteins.

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The effect of simple shear flow on the helix–coil transition of poly‐L‐lysine

Cited by 6 publications

References 0 publications

Do Protein Molecules Unfold in a Simple Shear Flow?

Do Protein Molecules Unfold in a Simple Shear Flow?

Synthesis and Conformational Transition of Surface-Tethered Polypeptide: Poly(l-lysine)

Rheo-NMR Studies of an Enzymatic Reaction: Evidence of a Shear-Stable Macromolecular System

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