Hydrophobic Core Malleability of a De Novo Designed Three-helix Bundle Protein

Walsh, Scott T. R.; Sukharev, V. I.; Betz, Stephen F.; Векшин, Н. Л.; DeGrado, William F.

doi:10.1006/jmbi.2000.4184

Cited by 36 publications

(45 citation statements)

References 56 publications

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“…11,23 ␣ 3 D itself has a somewhat lower heat capacity for folding than the average for natural proteins, indicative of slightly greater penetration of water molecules in the native state, 43 and the designed protein was more forgiving of core mutations than for most natural proteins. 44 All of these observations taken together strongly suggest that the reduced desolvation barrier and reduced frustration of less packed hydrophobic cores play important roles in facilitating downhill folding.…”

Section: Discussionmentioning

confidence: 97%

“…␣ 3 D shows most hallmarks of native proteins, but it is an early computational design, and it has been suggested that ␣ 3 D has a more malleable and flexible hydrophobic core than most evolved native proteins. 32 The flexibility of the hydrophobic core of ␣ 3 D lies between that of a native protein and a molten globule. 32 By lowering the pH of the protein solution from 5.0 ͑Ref.…”

Section: Discussionmentioning

confidence: 99%

“…32 The flexibility of the hydrophobic core of ␣ 3 D lies between that of a native protein and a molten globule. 32 By lowering the pH of the protein solution from 5.0 ͑Ref. 32͒ to 2.6 ͑present work and IR study 29 ͒, the flexibility of the hydrophobic core of ␣ 3 D could be tuned to be even more molten globule-like.…”

Section: Discussionmentioning

confidence: 99%

“…32 To make a direct comparison with the folding kinetics measurement monitored by infrared absorption spectroscopy of the amide IЈ band at 1631 and 1665 cm −1 , 29 the protein sample was purified and prepared in the same way as described in Ref. 29.…”

Section: Methodsmentioning

confidence: 99%

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A one-dimensional free energy surface does not account for two-probe folding kinetics of protein α3D

Liu

Dumont

Zhu

et al. 2009

The Journal of Chemical Physics

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We present fluorescence-detected measurements of the temperature-jump relaxation kinetics of the designed three-helix bundle protein ␣ 3 D taken under solvent conditions identical to previous infrared-detected kinetics. The fluorescence-detected rate is similar to the IR-detected rate only at the lowest temperature where we could measure it ͑326 K͒. The fluorescence-detected rate decreases by a factor of 3 over the 326-344 K temperature range, whereas the IR-detected rate remains nearly constant over the same range. To investigate this probe dependence, we tested an extensive set of physically reasonable one-dimensional ͑1D͒ free energy surfaces by Langevin dynamics simulation. The simulations included coordinate-and temperature-dependent roughness, diffusion coefficients, and IR/fluorescence spectroscopic signatures. None of these can reproduce the IR and fluorescence data simultaneously, forcing us to the conclusion that a 1D free energy surface cannot accurately describe the folding of ␣ 3 D. This supports the hypothesis that ␣ 3 D has a multidimensional free energy surface conducive to downhill folding at 326 K, and that it is already an incipient downhill folder with probe-dependent kinetics near its melting point.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A one-dimensional free energy surface does not account for two-probe folding kinetics of protein α3D

Liu

Dumont

Zhu

et al. 2009

The Journal of Chemical Physics

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“…In addition, de novo designed proteins seem to be more flexible and malleable than their natural counterparts. For example, NMR analysis of the structure of ␣ 3 D, a de novo designed three-helix-bundle protein, showed that the protein adjusted in different ways to either alanine to leucine or alanine to isoleucine substitutions at the same position in the hydrophobic core (29). Whereas the leucine mutant showed reduced dispersion of the amide backbone and methyl side-chain resonances, the isoleucine mutant yielded well defined chemical shift dispersions, an indicator of native core packing.…”

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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Design of a Three‐Helix Bundle Capable of Binding Heavy Metals in a Triscysteine Environment

et al. 2011

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An important objective of de novo protein design is the preparation of metalloproteins, as many natural systems contain metals that play crucial roles for the function and/or structural integrity of the biopolymer. [1,2] Metalloproteins catalyze some of the most important processes in nature, from energy generation and transduction to complex chemical transformations. At the same time, metals in excess can be deleterious to cells, and some ions are purely toxic, with no known beneficial effects (e.g., Hg II or Pb II ). Ideally, we would hope to be able to use an approach based on first principles to create both known metallocenters and novel sites, which may lead to exciting new catalytic transformations. However, the design of novel metalloproteins is a challenging and complex task, especially if the aim is to prepare asymmetric metal environments.Numerous metalloprotein systems have been designed over the past 15 years, typically through the use of unassociated peptides that assemble into three-stranded coiled coils or helix-loop-helix motifs that form antiparallel fourstranded bundles. In terms of metal-ion binding, these systems have been functionalized with heme [3,4] and nonheme mononuclear [5] and binuclear centers. [6,7] It is often difficult to prepare nonsymmetrical metal sites through these strategies owing to the symmetry of the systems, which rely on homooligomerization. Thus, the preparation of a single polypeptide chain capable of controlling a metal-coordination environment is a key objective.Previously, we designed soft, thiol-rich metal-binding sites involving cysteine and/or penicillamine as the ligating amino acid residues into the interior of parallel, three-stranded ahelical coiled coils. [8,9] These systems have served as hallmarks for understanding the metallobiochemistry of different heavy metals, such as Cd II , Hg II , As III , and Pb II . [8][9][10][11] We have shown how to control the geometry and coordination number of metals such as Cd II and Hg II at the protein interior and how to fine-tune the physical properties of the metals, which led to site-selective molecular recognition of Cd II . [12][13][14] Although these homotrimeric assemblies have been very useful, the production of heterotrimeric systems in which metal environments could be fine-tuned controllably or a hydrogen bond could be introduced site-specifically has been elusive. [15] Therefore, we chose an alternative strategy to satisfy this objective and used a single polypeptide chain instead of multiple self-associating peptides.Existing designed heteromeric helical bundles and coiled coils show energetic preferences of several kcal mol À1 for the desired heteromeric versus homomeric assemblies. [16,17] However, the energy gap between a hetero-and homomeric assembly often depends critically on ionic strength, the pH value, and other environmental parameters. Moreover, the objective of many studies in de novo protein design is to make the metal ion adopt an energetically suboptimal coordination geometry, and the degree t...

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Hydrophobic Core Malleability of a De Novo Designed Three-helix Bundle Protein

Cited by 36 publications

References 56 publications

A one-dimensional free energy surface does not account for two-probe folding kinetics of protein α3D

A one-dimensional free energy surface does not account for two-probe folding kinetics of protein α3D

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

Design of a Three‐Helix Bundle Capable of Binding Heavy Metals in a Triscysteine Environment

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