De novo protein design: from molten globules to native-like states

164

196

Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed de novo to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled-coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favorable system for probing details of the molecular origins of amyloid disease.

Section: Resultssupporting

confidence: 77%

Exploring amyloid formation by a de novo design

Kammerer

Kostrewa²,

Zurdo³

et al. 2004

164

196

“…Table 1 lists the melting temperature and the van't Hoff enthalpies of the systems analyzed. Small, single-domain globular proteins typically have unfolding enthalpies (⌬H m ) on the order of Ϸ40 kcal͞mol (5,6). The enthalpies that we have measured are somewhat lower than that (Ϸ10 -15 kcal͞mol), as expected, because our complexes have much less buried hydrophobic surface area.…”

Section: Resultsmentioning

confidence: 67%

“…Therefore, two challenges occur in the design of ''native-like'' proteins. By using positive design (3,(5)(6)(7), one can take into account issues such as hydrophobicity and the helix-or sheet-forming propensity of amino acids. By using negative design [a term developed to describe the selective introduction of unique stabilizing interactions (2,3)], one can introduce features that stabilize one specific fold, while, at the same time, they destabilize alternative topologies.…”

mentioning

confidence: 99%

De novodesigned cyclic-peptide heme complexes

Rosenblatt

Wang

Suslick

2003

The structural characterization of de novo designed metalloproteins together with determination of chemical reactivity can provide a detailed understanding of the relationship between protein structure and functional properties. Toward this goal, we have prepared a series of cyclic peptides that bind to water-soluble metalloporphyrins (Fe III and Co III ). Neutral and positively charged histidine-containing peptides bind with a high affinity, whereas anionic peptides bind only weakly to the negatively charged metalloporphyrin. Additionally, it was found that the peptide becomes helical only in the presence of the metalloporphyrin. CD experiments confirm that the metalloporphyrin binds specific cyclic peptides with high affinity and with isodichroic behavior. Thermal unfolding experiments show that the complex has ''native-like'' properties. Finally, NMR spectroscopy produced well dispersed spectra and experimental restraints that provide a high-resolution solution structure of the complexed peptide.O ne approach to understanding the folding and function of proteins is to attempt their design from first principles (1-3). Such design, however, requires not only incorporation of nonpolar interactions, but also inclusion of specific hydrogen bonds, disulfide bridges, ion pairs, and metal chelation. Without these interactions, proteins form compact folding intermediates referred to as molten globules (4). Molten-globule structures are compact and possess a high degree of secondary structure, but they are also highly dynamic, with both internal and external side chains rapidly interconverting among a large family of rotamers. Analysis of protein structures, on the other hand, reveals that the interior hydrophobic core is well packed and rigid. Therefore, two challenges occur in the design of ''native-like'' proteins. By using positive design (3, 5-7), one can take into account issues such as hydrophobicity and the helix-or sheet-forming propensity of amino acids. By using negative design [a term developed to describe the selective introduction of unique stabilizing interactions (2, 3)], one can introduce features that stabilize one specific fold, while, at the same time, they destabilize alternative topologies. By using this dual strategy, one can increase the free-energy gap between the native and molten-globule states.With the challenge of design making rapid progress, several groups have begun to look at the incorporation of functional cofactors in hopes of producing de novo catalytic proteins (8-14). Many enzymes require mono-and dinuclear metal ions (15), metal clusters (ferredoxin and nitrogenase) (16), heme (cytochromes and globins) (17), and organic cofactors (flavin-and quinone-based enzymes) (18). These cofactors are important, especially when processes such as oxidation͞reduction or substrate binding and activation are required (15).Studies of natural enzymes have led to considerable understanding of their structure and function. Reduction of these complicated systems to minimal models, however, will allow ...

“…Well-folded native proteins display thermodynamic and structural properties that distinguish them from molten globule folding intermediates (29). Thermodynamically, native proteins differ from molten globules by undergoing cooperative thermal denaturations with relatively large enthalpy changes.…”

Section: Discussionmentioning

confidence: 99%

Rationally designed mutations convertde novoamyloid-like fibrils into monomeric β-sheet proteins

Wang

Hecht

2002

162

Amyloid fibrils are associated with a variety of neurodegenerative maladies including Alzheimer's disease and the prion diseases. The structures of amyloid fibrils are composed of ␤-strands oriented orthogonal to the fibril axis (''cross ␤'' structure). We previously reported the design and characterization of a combinatorial library of de novo ␤-sheet proteins that self-assemble into fibrillar structures resembling amyloid. The libraries were designed by using a ''binary code'' strategy, in which the locations of polar and nonpolar residues are specified explicitly, but the identities of these residues are not specified and are varied combinatorially. The initial libraries were designed to encode proteins containing amphiphilic ␤-strands separated by reverse turns. Each ␤-strand was designed to be seven residues long, with polar (E) and nonpolar (F) amino acids arranged with an alternating periodicity (EFEFEFE). The initial design specified the identical polar͞nonpolar pattern for all of the ␤-strands; no strand was explicitly designated to form the edges of the resulting ␤-sheets. With all ␤-strands preferring to occupy interior (as opposed to edge) locations, intermolecular oligomerization was favored, and the proteins assembled into amyloid-like fibrils. To assess whether explicit design of edgefavoring strands might tip the balance in favor of monomeric ␤-sheet proteins, we have now redesigned the first and͞or last ␤-strands of several sequences from the original library. In the redesigned ␤-strands, the binary pattern is changed from EFEFEFE to EFEKEFE (K denotes lysine). The presence of a lysine on the nonpolar face of a ␤-strand should disfavor fibrillar structures because such structures would bury an uncompensated charge. The nonpolar 3 lysine mutations, therefore, would be expected to favor monomeric structures in which the EFEKEFE sequences form edge strands with the charged lysine side chain accessible to solvent. To test this hypothesis, we constructed several second generation sequences in which the central nonpolar residue of either the N-terminal ␤-strand or the C-terminal ␤-strand (or both) is changed to lysine. Characterization of the redesigned proteins shows that they form monomeric ␤-sheet proteins.