Early Hydrophobic Collapse of α1-Antitrypsin Facilitates Formation of a Metastable State: Insights from Oxidative Labeling and Mass Spectrometry

Stocks, Bradley B.; Sarkar, Anindya; Wintrode, Patrick L.; Konermann, Lars

doi:10.1016/j.jmb.2012.08.019

Cited by 23 publications

(32 citation statements)

References 55 publications

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“…Subsequently, the ␣/␤ domain folds, and, in one of the last folding steps, strands 4 and 5B are incorporated into the mainly ␤ domain. This order of events is in good agreement with available data on AAT-folding kinetics from hydrogen/ deuterium exchange coupled to MS (33), fast photochemical oxidation coupled to MS (34), and tryptophan fluorescence spectroscopy (32).…”

Section: Gō-model Simulations Of Inhibitory Serpin Folding To the Actsupporting

confidence: 90%

“…Experimental studies of the kinetics of AAT folding have mainly focused on WT AAT (32)(33)(34) with limited data on the kinetics of Z unfolding (92,93). The results of the BF simulations are in good agreement with data from kinetic and equilibrium AAT folding studies.…”

Section: Investigating the Effects Of Mutations On Serpin Folding Usisupporting

confidence: 55%

“…As a prototypical case study, we discuss how both classes of methods have been used to simulate the folding of the canonical inhibitory serpin ␣ 1 -antitrypsin (AAT) a 394-amino acid protein with folding times as long as tens of minutes (32)(33)(34). The interest in this specific protein resides in the fact that it has a topologically complex native structure, and the functional conformation is a kinetically trapped state, not the lowest free energy conformation (35)(36)(37)(38)(39).…”

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confidence: 99%

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Successes and challenges in simulating the folding of large proteins

Gershenson

Gosavi

Faccioli

et al. 2020

Journal of Biological Chemistry

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Computational simulations of protein folding can be used to interpret experimental folding results, to design new folding experiments, and to test the effects of mutations and small molecules on folding. However, whereas major experimental and computational progress has been made in understanding how small proteins fold, research on larger, multidomain proteins, which comprise the majority of proteins, is less advanced. Specifically, large proteins often fold via long-lived partially folded intermediates, whose structures, potentially toxic oligomerization, and interactions with cellular chaperones remain poorly understood. Molecular dynamics based folding simulations that rely on knowledge of the native structure can provide critical, detailed information on folding free energy landscapes, intermediates, and pathways. Further, increases in computational power and methodological advances have made folding simulations of large proteins practical and valuable. Here, using serpins that inhibit proteases as an example, we review native-centric methods for simulating the folding of large proteins. These synergistic approaches range from Gō and related structurebased models that can predict the effects of the native structure on folding to all-atom-based methods that include side-chain chemistry and can predict how disease-associated mutations may impact folding. The application of these computational approaches to serpins and other large proteins highlights the successes and limitations of current computational methods and underscores how computational results can be used to inform experiments. These powerful simulation approaches in combination with experiments can provide unique insights into how large proteins fold and misfold, expanding our ability to predict and manipulate protein folding.

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Section: Gō-model Simulations Of Inhibitory Serpin Folding To the Actsupporting

confidence: 90%

Section: Investigating the Effects Of Mutations On Serpin Folding Usisupporting

confidence: 55%

mentioning

confidence: 99%

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Successes and challenges in simulating the folding of large proteins

Gershenson

Gosavi

Faccioli

et al. 2020

Journal of Biological Chemistry

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“…Constraining the gate itself may also be important, as conformational changes in this region have been implicated in pathological serpin polymerization (26), which can occur in the ER during serpin maturation. For the serpins α 1 -antitrypsin and ovalbumin, both of which lack C-terminal disulfide bonds, purified protein-folding studies have demonstrated the importance of the C-terminal region for proper folding and function (27)(28)(29)(30). These results highlight the importance of constraining conformationally labile regions early in serpin folding to efficiently reach the functionally required metastable structure.…”

Section: Atiiimentioning

confidence: 99%

Cellular folding pathway of a metastable serpin

Chandrasekhar

Wang

et al. 2016

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

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Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly β-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the proteasebinding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes.

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“…Mayo and colleagues introduced a multiple‐mix microchannel quench flow system capable of H/D pulse labeling and quenching with a 110 µs time resolution . Another technique that shows great promise takes advantage of bottom‐up MS coupled with hydroxyl radical footprinting . Lapidus and colleagues have successfully interfaced this capability with a laminar‐flow mixer to obtain oxidative labeling of surface exposed residues during the folding of hen egg lysozyme .…”

Section: Recent Trends and Future Directionsmentioning

confidence: 99%

Advances in turbulent mixing techniques to study microsecond protein folding reactions

et al. 2013

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Recent experimental and computational advances in the protein folding arena have shown that the readout of the one-dimensional sequence information into three-dimensional structure begins within the first few microseconds of folding. The initiation of refolding reactions has been achieved by several means, including temperature jumps, flash photolysis, pressure jumps and rapid mixing methods. One of the most commonly used means of initiating refolding of chemically-denatured proteins is by turbulent flow mixing with refolding dilution buffer, where greater than 99% mixing efficiency has been achieved within 10’s of microseconds. Successful interfacing of turbulent flow mixers with complementary detection methods, including time-resolved Fluorescence Spectroscopy (trFL), Förster Resonance Energy Transfer (FRET), Circular Dichroism (CD), Small-Angle X-ray Scattering (SAXS), Hydrogen Exchange (HX) followed by Mass Spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (NMR), Infrared Spectroscopy (IR), and Fourier Transform IR Spectroscopy (FTIR), has made this technique very attractive for monitoring various aspects of structure formation during folding. Although continuous-flow (CF) mixing devices interfaced with trFL detection have a dead time of only 30 µs, burst-phases have been detected in this time scale during folding of peptides and of large proteins (e.g., CheY and TIM barrels). Furthermore, a major limitation of CF mixing technique has been the requirement of large quantities of sample. In this brief communication, we will discuss the recent flurry of activity in micromachining and microfluidics, guided by computational simulations, that are likely to lead to dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.

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Early Hydrophobic Collapse of α1-Antitrypsin Facilitates Formation of a Metastable State: Insights from Oxidative Labeling and Mass Spectrometry

Cited by 23 publications

References 55 publications

Successes and challenges in simulating the folding of large proteins

Successes and challenges in simulating the folding of large proteins

Cellular folding pathway of a metastable serpin

Advances in turbulent mixing techniques to study microsecond protein folding reactions

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