Artificial diiron proteins: From structure to function

Calhoun, Jennifer R.; Nastri, Flavia; Maglio, Ornella; Pavone, Vincenzo; Lombardi, Angela; DeGrado, William F.

doi:10.1002/bip.20230

Cited by 93 publications

(96 citation statements)

References 92 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Along with combinatorial library technology, a variety of screening systems, such as phage display, ribosome display, etc., have been constructed. [1][2][3][4][5] Although a large library around 10 12 can be prepared using phage and mRNA display systems, the number of all sequences of proteins using 20 amino acids at each position is too large to be covered. For example, the number of possible protein sequences resulting from 100 amino acid residues is 10 130 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Selection and structural analysis of de novo proteins from an α3β3 genetic library

et al. 2009

View full text Add to dashboard Cite

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding a3b3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP-based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8-anilino-1-naphthalenesulfonate (ANS) binding assay, and analytical size-exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both a-helix and b-sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten-globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three a-helices and three b-strands as intended by its design. Thus, it would appear that artificially generated a3b3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.

show abstract

Section: Introductionmentioning

confidence: 99%

“…DeGrado and co-workers successfully designed catalytic metalloproteins capable of catalyzing a phenol oxidase reaction. 11,12 These metalloproteins composed of a four-helix bundle structure were well-packed, and the catalytic residues were well organized in the hydrophobic core. M.H.…”

Section: Introductionmentioning

confidence: 99%

Selection and structural analysis of de novo proteins from an α3β3 genetic library

et al. 2009

View full text Add to dashboard Cite

show abstract

“…Early demonstrations showed iron sulfur clusters spontaneously assemble in maquettes 2 singly or in pairs, with or without heme(s); later singlechain variants were designed [Gibney et al, 1996;Mulholland et al, 1998;Musgrave et al, 2002;Nanda et al, 2005;Grzyb et al, 2010;Nanda et al, 2016]. Maquette frames of the kind in Figure 7 have also proved to offer a creative environment for metals alone and in clusters ( Figure 5C) [Dieckmann et al, 1997;Farrer et al, 2000;Calhoun et al, 2005;Geremia et al, 2005;Touw et al, 2007;Calhoun et al, 2008;Tegoni et al, 2012;Zastrow et al, 2012;Roy et al, 2014].…”

Section: First-principles Of De Novo Designed Protein Structures Outlmentioning

confidence: 99%

Maquette Strategy for Creation of Light- and Redox-Active Proteins

Ennist¹,

Mancini²,

Auman³

et al. 2017

Photosynthesis and Bioenergetics

View full text Add to dashboard Cite

Twenty five years ago we began utilizing the maquette strategy long used by sculptors and architects to develop first-principles de novo proteins that could perform functions inspired by natural proteins. This brief account traces our approach to engineering light-and redox-driven activities in structurally elementary maquette proteins that reflect little or no primary sequence relationship with those of the inspiring natural system. We follow first-principle maquette protein development from their original production by chemical synthesis of individual helix peptides with di-cystenyl-linked assembly into four-helix bundle proteins, to their bacterial expression as single chain maquette proteins and recently to cellular assembly with selected light-and redox-active cofactors. This last advance includes integration of expressed maquettes with the natural machinery of cofactor maturation and transmembrane transport and extends to maquette fusion with natural cofactor-equipped proteins in cells. Growing experience has led to the recognition that mechanistic components developed en route to one selected maquette function can be retained and applied to the engineering of maquettes designed for other activities. This flexibility plus extraordinary compatibility of maquette proteins with cellular operations explains the large number of maquettes currently in development directed toward performance of a broad range of light and redox driven functions in vitro and in vivo.

show abstract

“…Although the reactivity of these enzymes is quite diverse, there are common structural features both at the protein and at the metal site levels. DeGrado and Lombardi have conducted a careful retrostructural analysis of this class of proteins [122][123][124].…”

Section: Diiron Model Proteins: the Due-ferri Familymentioning

confidence: 99%