The Experts below are selected from a list of 1467 Experts worldwide ranked by ideXlab platform
Ivana Sirangelo - One of the best experts on this subject based on the ideXlab platform.
-
Glycation Accelerates Fibrillization of the Amyloidogenic
2016Co-Authors: Wfwf Apomyoglobin, Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F Apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F Apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F Apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F Apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of th
-
Glycation of Wild-Type Apomyoglobin Induces Formation of Highly Cytotoxic Oligomeric Species.
2015Co-Authors: Clara Iannuzzi, G. Irace, Vincenzo Carafa, Lucia Altucci, Margherita Borriello, Roberto Vinciguerra, Ivana SirangeloAbstract:Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F Apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type Apomyoglobin. Our results show that glycation covalently modifies Apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type Apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type Apomyoglobin have been also investigated. J. Cell. Physiol. 9999: 2807–2820, 2015. © 2015 Wiley Periodicals, Inc.
-
Glycation accelerates fibrillization of the amyloidogenic W7FW14F Apomyoglobin.
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F Apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F Apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F Apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F Apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.
-
Misfolding and Amyloid Aggregation of Apomyoglobin
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated Apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of Apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process.
-
www.mdpi.com/journal/ijms Misfolding and Amyloid Aggregation of Apomyoglobin
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Abstract: Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated Apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of Apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process
G. Irace - One of the best experts on this subject based on the ideXlab platform.
-
Glycation Accelerates Fibrillization of the Amyloidogenic
2016Co-Authors: Wfwf Apomyoglobin, Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F Apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F Apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F Apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F Apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of th
-
Glycation of Wild-Type Apomyoglobin Induces Formation of Highly Cytotoxic Oligomeric Species.
2015Co-Authors: Clara Iannuzzi, G. Irace, Vincenzo Carafa, Lucia Altucci, Margherita Borriello, Roberto Vinciguerra, Ivana SirangeloAbstract:Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F Apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type Apomyoglobin. Our results show that glycation covalently modifies Apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type Apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type Apomyoglobin have been also investigated. J. Cell. Physiol. 9999: 2807–2820, 2015. © 2015 Wiley Periodicals, Inc.
-
Glycation accelerates fibrillization of the amyloidogenic W7FW14F Apomyoglobin.
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F Apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F Apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F Apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F Apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.
-
Misfolding and Amyloid Aggregation of Apomyoglobin
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated Apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of Apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process.
-
www.mdpi.com/journal/ijms Misfolding and Amyloid Aggregation of Apomyoglobin
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Abstract: Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated Apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of Apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process
Clara Iannuzzi - One of the best experts on this subject based on the ideXlab platform.
-
Glycation Accelerates Fibrillization of the Amyloidogenic
2016Co-Authors: Wfwf Apomyoglobin, Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F Apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F Apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F Apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F Apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of th
-
Glycation of Wild-Type Apomyoglobin Induces Formation of Highly Cytotoxic Oligomeric Species.
2015Co-Authors: Clara Iannuzzi, G. Irace, Vincenzo Carafa, Lucia Altucci, Margherita Borriello, Roberto Vinciguerra, Ivana SirangeloAbstract:Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F Apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type Apomyoglobin. Our results show that glycation covalently modifies Apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type Apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type Apomyoglobin have been also investigated. J. Cell. Physiol. 9999: 2807–2820, 2015. © 2015 Wiley Periodicals, Inc.
-
Glycation accelerates fibrillization of the amyloidogenic W7FW14F Apomyoglobin.
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F Apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F Apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F Apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F Apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.
-
Misfolding and Amyloid Aggregation of Apomyoglobin
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated Apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of Apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process.
-
www.mdpi.com/journal/ijms Misfolding and Amyloid Aggregation of Apomyoglobin
2013Co-Authors: Clara Iannuzzi, G. Irace, Rosa Maritato, Ivana SirangeloAbstract:Abstract: Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated Apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of Apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process
Gaetano Irace - One of the best experts on this subject based on the ideXlab platform.
-
Aggregation in Wild-Type Protein In Vitro
2013Co-Authors: Amyloidogenic Wfwf Apomyoglobin, Silvia Vilasi, Rosa Maritato, Rosalba Sarcina, Antonella De Simone, Gaetano IraceAbstract:Glycosaminoglycans (GAGs) are frequently associated with amyloid deposits in most amyloid diseases, and there is evidence to support their active role in amyloid fibril formation. The purpose of this study was to obtain structural insight into GAGprotein interactions and to better elucidate the molecular mechanism underlying the effect of GAGs on the amyloid aggregation process and on the related cytotoxicity. To this aim, using Fourier transform infrared and circular diochroism spectroscopy, electron microscopy and thioflavin fluorescence dye we examined the effect of heparin and other GAGs on the fibrillogenesis and cytotoxicity of aggregates formed by the amyloidogenic W7FW14 Apomyoglobin mutant. Although this protein is unrelated to human disease, it is a suitable model for in vitro studies because it forms amyloid-like fibrils under physiological conditions of pH and temperature. Heparin strongly stimulated aggregation into amyloid fibrils, thereby abolishing the lag-phase normally detected following the kinetics of the process, and increasing the yield of fibrils. Moreover, the protein aggregates were harmless when assayed for cytotoxicity in vitro. Neutral or positive compounds did not affect the aggregation rate, and the early aggregates were highly cytotoxic. The surprising result that heparin induced amyloid fibril formation in wild-type Apomyoglobin and in the partially folded intermediate state of the mutant, i.e., proteins that normally do not show any tendency to aggregate, suggested that the interaction of heparin with Apomyoglobin is highly specific because of the presence, in protein turn regions, of consensus sequences consisting of alternating basic an
-
Glycation of the W7FW17F Apomyoglobin.
2013Co-Authors: Clara Iannuzzi, Rosa Maritato, Gaetano Irace, Ivana SirangeloAbstract:Protein glycation monitored by fluorescence spectroscopy. W7FW14F Apomyoglobin was incubated in the absence (white bar) and in the presence of 0.5 M D-ribose (grey bar) and 0.5 M D-glucose (black bar) and changes in maximal fluorescence intensity were monitored at different time intervals. Protein concentration was 40 µM, other experimental details are described in the Materials and Methods section. (A) Maximal fluorescence intensity recorded at 410 nm upon excitation at 320 nm. (B) Maximal fluorescence intensity recorded at 425 nm upon excitation at 370 nm. The average value (±SD) of a quadruplicate experiment is plotted.
-
Effect of heparin concentration on W7FW14F Apomyoglobin fibrillation kinetics.
2013Co-Authors: Silvia Vilasi, Rosa Maritato, Gaetano Irace, Rosalba Sarcina, Antonella De Simone, Ivana SirangeloAbstract:Fibrillization was monitored by the increase in fluorescence of ThT, as described under Materials and Methods. Protein concentration was 40 µM. Heparin concentrations were 0.1 (○), 0.06 (□), 0.015 (♦), 0.010 (▴), 0.003 (▪), and 0 mg/mL (•).The inset shows the dependence of the transition midpoint on heparin/Apomyoglobin molar ratio.
-
Effect of ribosylation on the amyloid formation for the W7FW17F Apomyoglobin.
2013Co-Authors: Clara Iannuzzi, Rosa Maritato, Gaetano Irace, Ivana SirangeloAbstract:W7FW14F Apomyoglobin (40 µM) was incubated in the absence (light gray bar) and in the presence of 0.5M D-ribose (dark grey bar) and aliquots of each sample at different incubation time intervals were monitored by ThT fluorescence. The average value (±SD) of a quadruplicate experiment is plotted.
-
Western blot of ribosylated W7FW17F Apomyoglobin.
2013Co-Authors: Clara Iannuzzi, Rosa Maritato, Gaetano Irace, Ivana SirangeloAbstract:W7FW14F Apomyoglobin was incubated in the absence and in the presence of 0.5M D-ribose and aliquots were taken at different time intervals. (A) Western blot analysis using an anti-myoglobin antibody. (B) Dot blot analysis using an anti AGE antibody. Experimental conditions are described in the Materials and Methods section.
Robert L. Baldwin - One of the best experts on this subject based on the ideXlab platform.
-
The unfolding enthalpy of the pH 4 molten globule of Apomyoglobin measured by isothermal titration calorimetry
2000Co-Authors: Marc Jamin, Stewart N. Loh, M. Antalik, D.w. Bolen, Robert L. BaldwinAbstract:The unfolding enthalpy of the pH 4 molten globule from sperm whale Apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native Apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.
-
trifluoroethanol stabilizes the ph 4 folding intermediate of sperm whale Apomyoglobin
1998Co-Authors: Robert L. BaldwinAbstract:Abstract 2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of Apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native Apomyoglobin.
-
trifluoroethanol stabilizes the ph 4 folding intermediate of sperm whale Apomyoglobin
1998Co-Authors: Yongzhang Luo, Robert L. BaldwinAbstract:2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of Apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native Apomyoglobin.
-
protonation behavior of histidine 24 and histidine 119 in forming the ph 4 folding intermediate of Apomyoglobin
1998Co-Authors: Bernhard H. Geierstanger, Marc Jamin, Brian F Volkman, Robert L. BaldwinAbstract:Heteronuclear NMR methods are used to study the protonation of histidine and aspartate residues in the acid-induced unfolding of recombinant sperm whale Apomyoglobin. The results are combined with fluorescence and circular dichroism measurements of acid-induced unfolding of wild-type and double mutant (H24V/H119F) proteins. They are consistent with a simple model in which the failure to protonate a single buried histidine, H24, is largely responsible for the partial unfolding of native (N) wild-type Apomyoglobin to the pH 4 folding intermediate (I). H24 is known to form an unusual interaction in which its side chain is buried and hydrogen-bonded to the side chain of H119. Two-dimensional 1H-15N heteronuclear NMR spectra indicate that H24 is present in the rare delta tautomeric form and remains neutral until N unfolds to I, while H119 becomes protonated before the N --> I reaction occurs. In the H24V/H119F double mutant, all histidines are protonated in N and the N --> I reaction occurs at lower pH. Therefore, the protonation of aspartate and/or glutamate residues must provide an additional driving force for the N to I reaction. Two-dimensional 1H-13C NMR experiments are used to measure the protonation of aspartates in selectively 13C-labeled Apomyoglobin; the results indicate that none of the aspartate residues has a strongly depressed pKa in N, as would be expected if it forms a stabilizing salt bridge.
-
Two forms of the pH 4 folding intermediate of Apomyoglobin
1998Co-Authors: Marc Jamin, Robert L. BaldwinAbstract:Abstract The pH 4 folding intermediate of Apomyoglobin exists in two forms (Ia, Ib) at equilibrium. Their ratio depends on pH, urea concentration and the presence or absence of a stabilizing anion (citrate, sulfate), and it does not depend on protein concentration. The Ia and Ib species are separated by a kinetic barrier and their interconversion can be monitored by tryptophan fluorescence in stopped-flow experiments. At pH 4.2, Ib is converted to Ia at low urea concentrations and urea unfolding gives the unfolding transition of Ia. During the refolding of native (N) Apomyoglobin at pH 6, starting from the acid unfolded species (U), both Ia and Ib appear as transient intermediates and both Ia and Ib appear as transient intermediates in the acid-induced unfolding of N. The results are consistent with a linear folding and unfolding pathway: U ⇌ Ia ⇌ Ib ⇌ N. Apomyoglobin provides the opportunity to investigate at equilibrium the structures and properties of two different kinetic folding intermediates. A non-obligatory dimeric species of the pH 4 intermediate is formed slowly and contributes to the refolding kinetics at concentrations above 5 μM. The dimer dissociates slowly and during refolding at pH 6 it forms N in a later time range than does the monomer.