scholarly journals Extension of a de novo TIM barrel with a rationally designed secondary structure element

2021 ◽  
Vol 30 (5) ◽  
pp. 982-989
Author(s):  
Jonas Gregor Wiese ◽  
Sooruban Shanmugaratnam ◽  
Birte Höcker
Keyword(s):  
Secondary Structure ◽  
De Novo ◽  
Secondary Structure Element ◽  
Tim Barrel

scholarly journals Extension of a de novo TIM barrel with a rationally designed secondary structure element

2020 ◽  
Author(s):  
Jonas Gregor Wiese ◽  
Sooruban Shanmugaratnam ◽  
Birte Höcker
Keyword(s):  
High Resolution ◽  
Secondary Structure ◽  
Protein Design ◽  
Building Block ◽  
Biochemical Characterization ◽  
De Novo ◽  
X Ray ◽  
Future Design ◽  
Catalytic Sites ◽  
Tim Barrel

SummaryThe ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recently published de novo design of a four-fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein we developed a design pipeline based on computational ab initio folding that solves a less complex sub-problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high-resolution X-ray structure for one variant and compare it to our design model. The successful extension of this robust TIM-barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.

Secondary Structure in de Novo Designed Peptides Induced by Electrostatic Interaction with a Lipid Bilayer Membrane

Langmuir ◽  
2010 ◽  
Vol 26 (9) ◽  
pp. 6437-6448 ◽  
Author(s):  
Patrik Nygren ◽  
Martin Lundqvist ◽  
Bo Liedberg ◽  
Bengt-Harald Jonsson ◽  
Thomas Ederth
Keyword(s):  
Secondary Structure ◽  
Lipid Bilayer ◽  
Electrostatic Interaction ◽  
De Novo ◽  
Bilayer Membrane ◽  
Lipid Bilayer Membrane

scholarly journals Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion

2020 ◽  
Vol 117 (48) ◽  
pp. 30362-30369
Author(s):  
Shane J. Caldwell ◽  
Ian C. Haydon ◽  
Nikoletta Piperidou ◽  
Po-Ssu Huang ◽  
Matthew J. Bick ◽  
...  
Keyword(s):  
Protein Design ◽  
Metal Binding ◽  
De Novo ◽  
Enzymatic Reaction ◽  
Symmetric Domain ◽  
Lanthanide Ions ◽  
Internal Cavity ◽  
Tim Barrel ◽  
Reaction Chambers ◽  
Modular Platform

De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry—a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel—such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts.

scholarly journals Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/β protein

2019 ◽  
Vol 32 (7) ◽  
pp. 317-329
Author(s):  
Matthew Gill ◽  
Michelle E McCully
Keyword(s):  
Molecular Dynamics ◽  
Secondary Structure ◽  
Surface Area ◽  
De Novo ◽  
Md Simulations ◽  
Solvent Accessible Surface Area ◽  
Hydrophobic Core ◽  
Design Strategies ◽  
Major Interest ◽  
Core Packing

Abstract Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase A2 (AYEwt). The mutations in these designed proteins were intended to increase hydrophobic core packing and inter-secondary-structure interactions. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations of AYEwt and three designed variants at both 25 and 100°C. Our MD simulations agreed with the relative experimental stabilities of the designs based on their secondary structure content, Cα root-mean-square deviation/fluctuation, and buried-residue solvent accessible surface area. Using a contact analysis, we found that the designs stabilize inter-secondary structure interactions and buried hydrophobic surface area, as intended. Based on our analysis, we designed three additional variants to test the role of helix stabilization, core packing, and a Phe → Met mutation on thermostability. We performed the additional MD simulations and analysis on these variants, and these data supported our predictions.

scholarly journals Features of the secondary structure of a protein molecule from powder diffraction data

2010 ◽  
Vol 66 (7) ◽  
pp. 756-761 ◽  
Author(s):  
Sebastian Basso ◽  
Céline Besnard ◽  
Jonathan P. Wright ◽  
Irene Margiolaki ◽  
Andrew Fitch ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Powder Diffraction ◽  
De Novo ◽  
Structural Information ◽  
Heavy Atom ◽  
Protein Molecule ◽  
Lattice Parameter ◽  
Egg White Lysozyme ◽  
Replacement Method ◽  
Isomorphous Replacement

Protein powder diffraction is shown to be suitable for obtainingde novosolutions to the phase problem at low resolutionviaphasing methods such as the isomorphous replacement method. Two heavy-atom derivatives (a gadolinium derivative and a holmium derivative) of the tetragonal form of hen egg-white lysozyme were crystallized at room temperature. Using synchrotron radiation, high-quality powder patterns were collected in which pH-induced anisotropic lattice-parameter changes were exploited in order to reduce the challenging and powder-specific problem of overlapping reflections. The phasing power of two heavy-atom derivatives in a multiple isomorphous replacement analysis enabled molecular structural information to be obtained up to approximately 5.3 Å resolution. At such a resolution, features of the secondary structure of the lysozyme molecule can be accurately located using programs dedicated to that effect. In addition, the quoted resolution is sufficient to determine the correct hand of the heavy-atom substructure which leads to an electron-density map representing the protein molecule of proper chirality.

scholarly journals Loop size optimization: a new mechanism for protein stabilization

2016 ◽  
Author(s):  
Alessia Ruggiero ◽  
Nicole Balasco ◽  
Luciana Esposito ◽  
Luigi Vitagliano
Keyword(s):  
Secondary Structure ◽  
De Novo ◽  
Protein Structures ◽  
Protein Stabilization ◽  
Statistical Analyses ◽  
Fine Tuning ◽  
Marginal Stability ◽  
Loop Size ◽  
Thermophilic Organisms ◽  
The Impact

Motivation One of the fundamental issues in both chemistry and biology is the identification of the structural determinants that dictate protein folding and stability. The decoding of the folding code of protein structures would have a major impact on native structure prediction and on de novo design. This task is particularly difficult to achieve. Unlike synthetic polymers, protein structures combine complexity, fine-tuning and marginal stability. Despite these difficulties, in recent years major progresses have been made. A very recent breakthrough in the field is represented by the discovery of Baker and colleagues that the juxtaposition of basic secondary structure elements (α-helices and β-strands) follows well-defined rules ( Koga et al., 2012 ) . These investigations identified three fundamental rules for the preferences of βℓβ (strand-loop-strand), αℓβ (helix-loop-strand) and βℓα (strand-loop-helix) structural motifs. In particular, it was shown that the chirality of βℓβ and the orientation of βℓα/αℓβ strongly depend on the loop size. In this framework, we evaluated the impact of these rules on protein structures isolated from either (hyper)thermophilic or mesophilic organisms. We used the thioredoxin (Trx) system to experimentally validate the results emerged from the statistical analyses. Methods Statistical surveys Our statistical survey was based on the analyses of different structural databases made of proteins isolated from mesophilic or thermophilic organisms by assuming that the proteins of thermophilic species were on average more stable than those isolated from mesophilic ones. The adherence of these proteins to the rules identified by Baker and coworkers was evaluated. Experiments Wild-type E. coli Trx and a series of ad-hoc mutants were expressed and purified. The stability of these proteins was evaluated by CD spectroscopy. The structure of these variants was determined by X-ray crystallography. Results The statistical analyses indicate that in proteins isolated from thermophilic organisms better adhere to the Baker rules through the optimization of the size of the loop connecting secondary structure elements ( Balasco et al., 2013 ) . We then experimentally validated this mechanism using the thioredoxin isolated from E.coli (EcTrx), a widely characterized protein that has been used as a model in a large number of investigations ( Esposito et al., 2012 , Ruggiero et al., 2009 ) . Comparative analyses of loop sizes between EcTrx and Trx isolated from hyperthermophiles suggested that the size loop connecting helix 1 (α1) to strand 2 (β2) in EcTrx could be modified to better follow the rules. Chimeric variants were therefore prepared by replacing the loop of EcTrx with the corresponding ones present in the Trx isolated from Sulfolobus solfataricus and S. tokodaii. Abstract truncated at 3,000 characters - the full version is available in the pdf file

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