High Hydrostatic Pressure Can Reverse Aggregation of Protein Folding Intermediates and Facilitate Acquisition of Native Structure

Gorovits, Boris; Horowitz, Paul M.

doi:10.1021/bi9730137

Cited by 98 publications

(80 citation statements)

References 17 publications

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“…HHP already of pressures as low as 3.5 kbar is sufficient to weaken and (at least partially) disrupt the hydrophobic cores thus leading to formation of a heterogeneous population of fibrillar aggregates with IR amide I' bands in the low wave number region (which is typical of a more strongly H-bonding pattern of intermolecular β-sheets) and a large amount of non-fibrillar smaller aggregates and oligomers, as detected by AFM (with IR amide I' bands in the larger wave number region around 1620 cm -1 ). ii) Our data also indicate that the preformed IAPP fibrils are sensitive to high hydrostatic pressure, similar to amorphous aggregates and inclusion bodies [45][46][47]. Considering the fact that high hydrostatic pressure is an effective means in disturbing ionic and hydrophobic interactions but not hydrogen bonds, we can conclude that these former two types of interaction are important for the stability of IAPP fibrillar aggregates, as also suggested in work using denaturing agents [13].…”

Section: Discussionsupporting

confidence: 81%

Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

Lopes

Smirnovas

Winter

2008

J. Phys.: Conf. Ser.

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Section: Discussionsupporting

confidence: 81%

Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

Lopes

Smirnovas

Winter

2008

J. Phys.: Conf. Ser.

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“…The pressure-induced aggregation of rhIFN-␥ may seem counterintuitive, because pressure often dissociates multimeric proteins (27,(33)(34)(35)(36). However, rhIFN-␥ aggregates because the transition state, N*, has a smaller V i than does N. Thus, the equilibrium favors N* at higher pressures, increasing the rate of formation of M (step A) and accelerating aggregation.…”

Section: Discussionmentioning

confidence: 99%

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

Webb

Cleland

et al. 2001

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

106

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The equilibrium dissociation of recombinant human IFN-␥ was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was ؊209 ؎ 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 ؎ 1.6 nm 2 /molecule of dimer. The first-order aggregation rate of recombinant human IFN-␥ in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were ؊41 ؎ 9 ml/mol of dimer and 3.5 ؎ 0.2 nm 2 / molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values Ϸ4-fold larger for both transitions.A ggregation is of considerable concern to the medical, pharmaceutical, and biotechnology industries, as protein aggregation occurs in human diseases (1-4) and during the production, purification, and storage of protein products (5). Characterization of the conformational state(s) that lead(s) to aggregation is essential for a mechanistic understanding of the aggregation process.Protein aggregation was once viewed as a nonspecific hydrophobically driven process involving the fully unfolded random coil (6-8). However, recent research has shown protein aggregation to follow distinct pathways involving folding intermediates (6, 7, 9). Thus, the folding pathway and aggregation processes are critically linked (7,8). Characterization of the unfolding process, in conjunction with aggregation rate studies, should then provide insight into the mechanisms of protein aggregation and the role of folding intermediates therein. A major challenge in characterizing aggregation pathways is that under conditions that greatly favor the native state (e.g., physiological), the small populations of highly reactive aggregationcompetent species and the transition states leading to these species may be inaccessible to available spectroscopic techniques. To populate putative aggregation-competent species at levels sufficient for spectroscopic measurement, conditions that greatly perturb the native state are typically used (e.g., pH Ͻ4.0). Furthermore, even if the aggregation-competent species populated under nonn...

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“…For example, we have recently shown (48,49) that the surface area and volume changes required to form an aggregation competent species of recombinant human interferon-␥ corresponds to that expected for formation of a molten globule intermediate. Because aggregation may be inhibited under pressure (25), eliminating interference from aggregate signals, we suggest that high pressure spectroscopy may be of particular value for structural studies of molten globules induced either by high pressures or solution composition.…”

Section: Thermodynamics Of Pressure-induced Disaggregation Andmentioning

confidence: 99%

“…Gorovits and Horowitz (25) reported that high pressure could arrest protein aggregation under solution conditions that, at atmospheric pressure, favor formation of aggregates. Recently we have developed high pressure methods for disaggregating and refolding proteins from aggregates and inclusion bodies, allowing processing of high concentrations of protein (e.g.…”

mentioning

confidence: 99%

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

John¹,

Carpenter²,

Balny³

et al. 2001

Journal of Biological Chemistry

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Two different types of insoluble, non-native aggregates of recombinant human growth hormone were formed by agitation in buffer or buffer containing 0.75 M guanidine HCl (GdnHCl) and characterized by infrared and second derivative UV spectroscopies. The degree of secondary structural perturbation was greater in the aggregates formed in 0.75 M GdnHCl. Both aggregate types were dissolved and refolded using high hydrostatic pressures in combination with either elevated temperature or non-denaturing levels of guanidine HCl or urea. The effects of a range of temperature, pressure, and chaotrope concentrations were tested and led to optimal conditions that approached 100% yield of native protein. The aggregates formed in 0.75 M GdnHCl required higher concentrations of urea or GdnHCl, or higher temperature or pressure for a yield equivalent to that for aggregates formed in buffer alone. Investigation of the effects of pressure, temperature, and chaotrope on unfolding of rhGH documented that under conditions used for optimal high pressure disaggregation and refolding, the native state is greatly favored thermodynamically (e.g. 25 kJ/mol at 2000 bar and 0.75 M GdnHCl). Dissolution of aggregates under pressure is a kinetically limited process. Comparison of refolding yields in GdnHCl and urea solutions suggest that pressure effects on electrostatic interactions do not dominate pressure effects on disaggregation. We suggest that non-native hydrogen bonds between protein molecules within aggregates of recombinant human growth hormone are responsible for the rate-limiting kinetic barrier in pressure-induced disaggregation.

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High Hydrostatic Pressure Can Reverse Aggregation of Protein Folding Intermediates and Facilitate Acquisition of Native Structure

Cited by 98 publications

References 17 publications

Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

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