Fluorescence-detected polymerization kinetics of human α1-antitrypsin

Kołoczek, Henryk; Guz, Andrzej; Kaszycki, Paweł

doi:10.1007/bf01886851

Cited by 12 publications

(10 citation statements)

References 21 publications

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“…2). Although previous studies (8) have found that α 1 -AT will polymerize upon incubation in low concentrations of denaturant, this process showed a strong temperature dependence. Refolding conditions in the present study employ both lower GuHCl concentrations (0.6 M after dilution as opposed to 1 M in Koloczek et al,ref.…”

Section: Resultsmentioning

confidence: 70%

Folding mechanism of the metastable serpin α ₁ -antitrypsin

Tsutsui

Cruz

Wintrode

2012

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

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The misfolding of serpins is linked to several genetic disorders including emphysema, thrombosis, and dementia. During folding, inhibitory serpins are kinetically trapped in a metastable state in which a stretch of residues near the C terminus of the molecule are exposed to solvent as a flexible loop (the reactive center loop). When they inhibit target proteases, serpins transition to a stable state in which the reactive center loop forms part of a six-stranded β-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human α 1 -antitrypsin (α 1 -AT). We find large differences in the folding kinetics of different regions. A key region in the metastable → stable transition, β-strand 5A, shows a lag phase of nearly 350 s. In contrast, the "B-C barrel" region shows no lag phase and the incorporation of the C-terminal residues into β-sheets B and C is largely complete before the center of β-sheet A begins to fold. We propose this as the mechanism for trapping α 1 -AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which α 1 -AT avoids polymerization during folding.hydrogen exchange | misfolding disease | protein folding T he misfolding and polymerization of serpins is linked to a number of inherited diseases including liver cirrhosis, emphysema, and dementia (1). In the most common serpin-linked disease, α 1 -antitrypsin deficiency, α 1 -antitrypsin (α 1 -AT) is trapped in a misfolded polymerization prone state during processing in the endoplasmic reticulum. Accumulation of polymers ultimately leads to apoptosis. Unlike in amyloid diseases, however, it is not thought that serpins undergo a compete change in their secondary and tertiary structures upon polymerization. Instead, serpin misfolding is thought to be related to their unusual inhibitory mechanism.The lowest free energy structure of α 1 -AT is shown in Fig. 1B. However, the structure shown in Fig. 1A is the structure that α 1 -AT spontaneously adopts during folding. Alpha-1 antitrypsin, like other inhibitory serpins, is trapped in a metastable state (2). The α 1 -AT fold consists of three β-sheets (A, B, and C) and nine α-helices (A-I). In the metastable form, residues 345-360 are exposed to solvent and comprise the reactive center loop (RCL) that is cleaved by target proteases, whereas in the stable form, these same residues form the fourth strand of β-sheet A. Cleavage by target proteases triggers a conformational change in which the N-terminal portion of the RCL spontaneously inserts into β-sheet A, carrying the bound protease (covalently linked as an acylenzyme intermediate) along with it.Evidence suggests that serpin polymerization is an intermolecular version of the above-described intramolecular process. The two most prominent models for serpin polymerization propose that polymers are formed either through the insertion of the RCL of one serpin into the A β-sheet of another, or through a domain swap mechani...

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Section: Resultsmentioning

confidence: 70%

Folding mechanism of the metastable serpin α ₁ -antitrypsin

Tsutsui

Cruz

Wintrode

2012

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

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“…[17][18][19] Why the Z mutation leads to increased polymerization of α 1 AT and which role the alternative native conformation plays during its polymerization process are still not clear though. In vitro studies suggest that wildtype and Z α 1 AT polymerize from the folding intermediate (I) 5,18,20 and that the Z mutation slows the final folding step, thus accumulating I. 21 This might explain why, in the cell, Z α 1 AT polymers are retained in the ER.…”

Section: Introductionmentioning

confidence: 98%

Structural Change in β-Sheet A of Z α1-Antitrypsin Is Responsible for Accelerated Polymerization and Disease

Knaupp

Bottomley

2011

Journal of Molecular Biology

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“…Studies of the un/folding pathways of wild type α 1 AT [4], [5], [6], [7], [8], [9], and Z α 1 AT [10], [11], [12], have indicated that both proteins follow a three-state pathway with the formation of a single intermediate ensemble (I) readily populated in approximately 1 M guanidine hydrochloride (GdnHCl). There is evidence that this folding intermediate of α 1 AT is prone to polymerization [13], [14], [15] and that the Z mutation leads to retardation of the second folding transition and an increased rate of unfolding from the native state and hence accumulation of the aggregation-prone species [11], [12].…”

Section: Introductionmentioning

confidence: 99%

The Roles of Helix I and Strand 5A in the Folding, Function and Misfolding of α1-Antitrypsin

et al. 2013

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α1-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α1-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α1-Antitrypsin has never been examined. We have therefore created a range of α1Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α1-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states.

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Fluorescence-detected polymerization kinetics of human α1-antitrypsin

Cited by 12 publications

References 21 publications

Folding mechanism of the metastable serpin α ₁ -antitrypsin

Folding mechanism of the metastable serpin α ₁ -antitrypsin

Structural Change in β-Sheet A of Z α1-Antitrypsin Is Responsible for Accelerated Polymerization and Disease

The Roles of Helix I and Strand 5A in the Folding, Function and Misfolding of α1-Antitrypsin

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Fluorescence-detected polymerization kinetics of human α1-antitrypsin

Cited by 12 publications

References 21 publications

Folding mechanism of the metastable serpin α 1 -antitrypsin

Folding mechanism of the metastable serpin α 1 -antitrypsin

Structural Change in β-Sheet A of Z α1-Antitrypsin Is Responsible for Accelerated Polymerization and Disease

The Roles of Helix I and Strand 5A in the Folding, Function and Misfolding of α1-Antitrypsin

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Folding mechanism of the metastable serpin α ₁ -antitrypsin

Folding mechanism of the metastable serpin α ₁ -antitrypsin