Folding of a de Novo Designed Native-like Four-helix Bundle Protein

Chapeaurouge, Alex; Johansson, Jonas; Ferreira, Sérgio T.

doi:10.1074/jbc.m105232200

Cited by 36 publications

(32 citation statements)

References 28 publications

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“…Thus, the most likely interpretation is that binding of bis-ANS occurred to a hydrophobic region located somewhere close to the active site but that this region is separate from the region engaged in binding of LPL to the lipid/ water interface of emulsified substrates. (47)(48)(49). In some cases bis-ANS may interact with specialized ligand-binding regions of native proteins.…”

Section: Studies Of the Complexes Of Bis-ans With Lpl By Circularmentioning

confidence: 99%

1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII

Lóokene

Zhang

Tõugu

et al. 2003

Journal of Biological Chemistry

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Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII),and a slow off-rate with a dissociation rate constant k diss ‫؍‬ 1.2 ؋ 10 ؊4 s ؊1 . The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII. Lipoprotein lipase (LPL)1 is one of the central proteins in blood lipid metabolism (for reviews, see Refs. 1-3). The enzyme is bound to heparan sulfate proteoglycans at the vascular endothelium and hydrolyzes triacylglycerols and phospholipids in plasma lipoproteins so that lipolysis products can be taken up in adjacent cells for metabolic purposes. In addition and independent of catalysis, the LPL protein functions as a mediator for binding and uptake of lipoproteins by cells by bridging between the lipoproteins and heparan sulfate proteoglycans or receptors of the low density lipoprotein receptor family.LPL belongs to the family of mammalian triglyceride lipases together with pancreatic lipase, hepatic lipase, and endothelial lipase (2). Based on sequence homology with pancreatic lipase, models of the 55-kDa subunit of LPL have been created (4 -6). The active form of LPL is a noncovalent dimer of identical subunits (7,8) that are arranged in a head-to-tail fashion (5, 9). LPL can engage in a number of interactions with heparin/ heparan sulfate, receptors, lipid/water interfaces, the activator apolipoprotein CII (apoCII), and perhaps with other apolipoproteins. Fatty acids with long acyl chains have been shown to bind to LPL with high affinity (10) and to inhibit the catalytic activity of LPL as well as binding of LPL to lipid/water interfaces (lipoproteins, emulsion droplets) (11) and to heparin (12). Thus, fatty acids exert strong control over the activity of LPL. Their removal by uptake in tissues or by binding to albumin is essential to allow the action of the enzyme to proceed unabated.ApoCII, a 79-amino acid residue peptide belonging to the apoC family of exchangeable apolipoproteins, is a necessary activator for LPL in vivo (13,14). Deficiency of apoCII leads to massive accumulation of lipids in blood and symptoms similar to what is seen on LPL deficiency (15). Systematic mutagenesis of residues in the LPL binding domain of apoCII and studies of the three-dimensional structure of apoCII by NMR strongly suggest that the basis for the activation is formation of a complex between apoCII and LPL at the surface of a lipid droplet (16,17). Although the region of apoCII responsible for the interaction with LPL has been localized to the C-terminal part of the peptide (18), it is still unclear which part(s) of LPL interacts wit...

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Section: Studies Of the Complexes Of Bis-ans With Lpl By Circularmentioning

confidence: 99%

1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII

Lóokene

Zhang

Tõugu

et al. 2003

Journal of Biological Chemistry

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“…The band intensity of the turn structure seems to slightly decrease as pressure increases. Recently, Chapeaurouge et al 20 reported the pressure-induced unfolding of ( 2 ) 2 four-helix bundle protein, which has nearly the same primary structure constructing the hydrophobic core of (-l-) 2 , using the spectral center of mass of Trp fluorescence. They concluded that the pressure of about 350 MPa induces reversible dissociation of dimmer of ( 2 ) 2 to partially folded monomer.…”

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confidence: 99%

Pressure stability of the α‐helix structure in a de novo designed protein (α‐l‐α)₂ studied by FTIR spectroscopy

Takekiyo

Takeda

Isogai³

et al. 2006

Biopolymers

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The pressure‐induced structural changes of a de novo designed four‐helix bundle protein, (α‐l‐α)2, in aqueous solution have been investigated by FTIR spectroscopy. Changes in the amide I' band intensity show that pressure induces disruption of tertiary interactions and stabilizes the solvated α‐helical form. This may suggest that the exposure of the hydrophobic core to the solvent by pressure is not a sufficient condition for pressure‐induced unfolding of the α‐helices of proteins. © 2006 Wiley Periodicals, Inc. Biopolymers 85:185–188, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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“…30 and 31) and de novo designed proteins (7,14,32). Hydrophobic interactions, which are believed to be the major stabilizing forces of protein cores, are destabilized at low temperatures, leading to protein unfolding (33).…”

Section: Discussionmentioning

confidence: 99%

Redesigning the Folding Energetics of a Model Three-helix Bundle Protein by Site-directed Mutagenesis

Lopes

Chapeaurouge

Manderson

et al. 2004

Journal of Biological Chemistry

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Because of their limited size and complexity, de novo designed proteins are particularly useful for the detailed investigation of folding thermodynamics and mechanisms. Here, we describe how subtle changes in the hydrophobic core of a model three-helix bundle protein (GM-0) alter its folding energetics. To explore the folding tolerance of GM-0 toward amino acid sequence variability, two mutant proteins (GM-1 and GM-2) were generated. In the mutants, cavities were created in the hydrophobic core of the protein by either singly (GM-1; L35A variant) or doubly (GM-2; L35A/I39A variant) replacing large hydrophobic side chains by smaller Ala residues. The folding of GM-0 is characterized by two partially folded intermediate states exhibiting characteristics of molten globules, as evidenced by pressureunfolding and pressure-assisted cold denaturation experiments. In contrast, the folding energetics of both mutants, GM-1 and GM-2, exhibit only one folding intermediate. Our results support the view that simple but biologically important folding motifs such as the threehelix bundle can reveal complex folding plasticity, and they point to the role of hydrophobic packing as a determinant of the overall stability and folding thermodynamic of the helix bundle.

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Folding of a de Novo Designed Native-like Four-helix Bundle Protein

Cited by 36 publications

References 28 publications

1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII

1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII

Pressure stability of the α‐helix structure in a de novo designed protein (α‐l‐α)₂ studied by FTIR spectroscopy

Redesigning the Folding Energetics of a Model Three-helix Bundle Protein by Site-directed Mutagenesis

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Folding of a de Novo Designed Native-like Four-helix Bundle Protein

Cited by 36 publications

References 28 publications

1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII

1,1′-Bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate Acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII

Pressure stability of the α‐helix structure in a de novo designed protein (α‐l‐α)2 studied by FTIR spectroscopy

Redesigning the Folding Energetics of a Model Three-helix Bundle Protein by Site-directed Mutagenesis

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Pressure stability of the α‐helix structure in a de novo designed protein (α‐l‐α)₂ studied by FTIR spectroscopy