Kinetics of Folding of the All-β Sheet Protein Interleukin-1β

Varley, Paul; Gronenborn, Angela M.; Christensen, Hans Erik Mølager; Wingfield, Paul T.; Rh, Pain; Clore, G. Marius

doi:10.1126/science.8493553

Cited by 170 publications

(148 citation statements)

References 22 publications

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“…51 Thus, our analysis of the folding of FGF-1 is inconsistent with the published report of Yu and coworkers, 48 but is in general agreement with the folding studies of other b-trefoil proteins in which the central strands of the protein are observed to fold faster than those on the periphery. 47,52,53 Only a key subset of structural elements (turns #2-#5 and turn #7, spanning $50% of the overall protein) appears necessary to confer efficient foldability to FGF-1. The entire protein is not (and apparently does not need to be) optimized for foldability; the regions not contributing to formation of the folding transition state instead segregate to regions of HSPG and receptor-binding functionalities.…”

Section: Discussionmentioning

confidence: 99%

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

2012

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The acquisition of function is often associated with destabilizing mutations, giving rise to the stability-function tradeoff hypothesis. To test whether function is also accommodated at the expense of foldability, fibroblast growth factor-1 (FGF-1) was subjected to a comprehensive u-value analysis at each of the 11 turn regions. FGF-1, a b-trefoil fold, represents an excellent model system with which to evaluate the influence of function on foldability: because of its threefold symmetric structure, analysis of FGF-1 allows for direct comparisons between symmetryrelated regions of the protein that are associated with function to those that are not; thus, a structural basis for regions of foldability can potentially be identified. The resulting u-value distribution of FGF-1 is highly polarized, with the majority of positions described as either foldedlike or denatured-like in the folding transition state. Regions important for folding are shown to be asymmetrically distributed within the protein architecture; furthermore, regions associated with function (i.e., heparin-binding affinity and receptor-binding affinity) are localized to regions of the protein that fold after barrier crossing (late in the folding pathway). These results provide experimental support for the foldability-function tradeoff hypothesis in the evolution of FGF-1. Notably, the results identify the potential for folding redundancy in symmetric protein architecture with important implications for protein evolution and design.

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Section: Discussionmentioning

confidence: 99%

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

2012

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“…4B). ␤-Strands 6 -10 in IL-1␤ (trefoil 2 and the leading edge of trefoil 3) were protected first during protein refolding as detected by pulse labeling NMR (22). These strands comprise an on-route folding intermediate of IL-1␤ (23,50).…”

Section: The C-terminal Region Of Pro-il-1␤ (Residues 117-269) Displamentioning

confidence: 99%

“…1B) (21). Mature IL-1␤ refolds relatively slowly (22), accessing multiple routes including a major route with a detectable intermediate population (23,24). Recently, this slow folding has been attributed to repacking of a functionally important loop (the ␤-bulge) in the mature protein (see Fig.…”

mentioning

confidence: 99%

Pro-interleukin (IL)-1β Shares a Core Region of Stability as Compared with Mature IL-1β While Maintaining a Distinctly Different Configurational Landscape

Hailey¹,

Andersen³

et al. 2009

Journal of Biological Chemistry

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Interleukin-1␤ (IL-1␤) is a master cytokine involved in initiating the innate immune response in vertebrates (Dinarello, C. A. (1994) FASEB J. 8, 1314 -1325). It is first synthesized as an inactive 269-residue precursor (pro-interleukin-1␤ or pro-IL-1␤). Pro-IL-1␤ requires processing by caspase-1 to generate the active, mature 153-residue cytokine. In this study, we combined hydrogen/deuterium exchange mass spectrometry, circular dichroism spectroscopy, and enzymatic digestion comparative studies to investigate the configurational landscape of pro-IL-1␤ and the role the N terminus plays in modulating the landscape. We find that the N terminus keeps pro-IL-1␤ in a protease-labile state while maintaining a core region of stability in the C-terminal region, the eventual mature protein. In mature IL-1␤, this highly protected region maps back to the area protected earliest in the NMR studies characterizing an on-route kinetic refolding intermediate. This protected region also encompasses two important functional loops that participate in the IL-1␤/receptor binding interface required for biological activity. We propose that the purpose of the N-terminal precursor region in pro-IL-1␤ is to suppress the function of the eventual mature region while keeping a structurally and also functionally important core region primed for the final folding into the native, active state of the mature protein. The presence of the self-inhibiting precursor region provides yet another layer of regulation in the life cycle of this important cytokine.Nearly all cell types respond to interleukin (IL)-1␤, 4 in a very sensitive manner, via binding to the interleukin-1 receptor type 1 (IL-1RI) (2). Although essential in the immune response, overproduction of IL-1␤ can lead to both acute (sepsis) as well as chronic (rheumatoid arthritis, atherosclerosis, obesity, and diabetes) disease states (3). Thus, the expression, activation, and secretion of this cytokine are tightly controlled (4). Although many cell types express IL-1␤, it is predominately produced and secreted by monocytes and macrophages (1). The protein is synthesized as a biologically inactive 269-residue precursor molecule, pro-interleukin-1␤ (pro-IL-1␤), and the 153-residue active mature IL-1␤ is generated from the C-terminal domain. Processing of the proprotein involves the recently discovered NALP-1 and NALP-3 inflammasomes, which are responsible for activating procaspase-1 (5). The inflammasome function is integral in wound repair as well as for combating infection (6 -9).In vivo, the 31-kDa pro-IL-1␤ precursor is processed to the active C-terminal 17-kDa form by the interleukin-1 converting enzyme, caspase-1 (10, 11). Caspase-1 is a cysteine protease that recognizes two cleavage sites in pro-IL-1␤, the Asp 27 2Gly 28 and Asp 116 2Ala 117 peptide bonds (Fig. 1A). These cleavage sites are conserved across mammals (12-14). The activation pathway is believed to proceed with cleavage first at Asp 27 2Gly 28 (site 1) followed by Asp 116 2Ala 117 (site 2). These processing events lead ...

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“…On the other hand, when using pulsed proton exchange followed by NMR identification of the protected protons, the formation of stable secondary structure elements could be detected over a slower observable time range (8,9). Using both techniques to observe the folding of the same protein, as was the case for cytochrome c (7), lysozyme (10,11), and interleukin-1␤ (12), secondary structures could be observed by CD at a stage where pulsed proton exchange/NMR failed to detect protected protons. This apparent contradiction suggested that secondary structure elements could form without providing an efficient protection against proton exchange.…”

mentioning

confidence: 99%

Kinetics of Secondary Structure Recovery during the Refolding of Reduced Hen Egg White Lysozyme

Roux¹,

Delepierre²,

Goldberg³

et al. 1997

Journal of Biological Chemistry

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We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 M oxidized glutathione, and 20 M reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi-or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.Since Anfinsen's work on the in vitro renaturation of unfolded ribonuclease A (1), it is commonly accepted that all the information required for a protein to fold properly is contained in its amino acid sequence. However, the code that allows the formation of a fully folded protein from its amino acid sequence has not yet been deciphered. Three models are currently proposed to describe this process. The framework model is a sequential model in which secondary structure elements form first followed by a tighter packing of the molecule (2). Another model assumes that the polypeptide chain undergoes a rapid collapse driven by hydrophobic forces that would yield an intermediate close to the molten globule (3). In the puzzle model (4) structural elements form at different sites on the polypeptide chain, and their formation induces further folding of the whole protein.According to experimental data, it is not yet possible to determine which model most accurately describes the folding mechanism. However, some general features of protein folding have arisen. Stopped-flow circular dichroism studies showed that a large amount of secondary structure is formed in the dead time (milliseconds time range) of the observation. This has been observed for proteins such as ␣-lactalbumin and lysozyme (5), dihydrofolate reductase (6), and holocytochrome c (7). On the other hand, when using pulsed proton exchange followed by NMR identification of the protected protons, the formation of stable secondary structure elements could be detected over a slower observable time range (8, 9). Using both techniques to observe the folding of the same protein, as was the case for cytochrome c (7), lysozyme (10, 11), and interleukin-1␤ (12), secondary structures coul...

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Kinetics of Folding of the All-β Sheet Protein Interleukin-1β

Cited by 170 publications

References 22 publications

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

Pro-interleukin (IL)-1β Shares a Core Region of Stability as Compared with Mature IL-1β While Maintaining a Distinctly Different Configurational Landscape

Kinetics of Secondary Structure Recovery during the Refolding of Reduced Hen Egg White Lysozyme

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