Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments

Autoren

Gianluca Interlandi
Svava K Wetzel
Giovanni Settanni
Andreas Plückthun
Amedeo Caflisch

DOI

10.1016/j.jmb.2007.09.042

ISSN

0022-2836

Ausgabe der Veröffentlichung

3

Zeitschrift

Journal of Molecular Biology

Sprache

en

Paginierung

837 - 854

Datum der Veröffentlichung

2008

Status

Published

Herausgeber

Elsevier BV

Herausgeber URL

http://dx.doi.org/10.1016/j.jmb.2007.09.042

Datum der Datenerfassung

2024

Titel

Characterization and Further Stabilization of Designed Ankyrin Repeat Proteins by Combining Molecular Dynamics Simulations and Experiments

Ausgabe der Zeitschrift

375

Datenquelle: Crossref

Abstract

Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations. Notably, a stable intermediate is found in experimental equilibrium unfolding studies of one of the simulated consensus proteins. On the basis of simulation results, this intermediate is interpreted to represent a conformation with a denatured C-terminal repeat. To validate this interpretation, constructs without C-terminal capping repeat were prepared and did not show this intermediate in equilibrium unfolding experiments. Conversely, the capping repeats were found to be essential for efficient folding in the cell and for avoiding aggregation, presumably because of their highly charged surface. To design a capping repeat conferring similar solubility properties yet even higher stability, eight point mutations adapting the C-cap to the consensus AR and adding a three-residue extension at the C-terminus were predicted in silico and validated experimentally. The in vitro full-consensus proteins were also compared with a previously published designed AR protein, E3_5, whose internal repeats show 80% identity in primary sequence. A detailed analysis of the simulations suggests that networks of salt bridges between beta-hairpins, as well as additional interrepeat hydrogen bonds, contribute to the extraordinary stability of the full consensus.

Addresses

Department of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland.

Autoren

Gianluca Interlandi
Svava K Wetzel
Giovanni Settanni
Andreas Plückthun
Amedeo Caflisch

DOI

10.1016/j.jmb.2007.09.042

eISSN

1089-8638

Externe Identifier

PubMed Identifier: 18048057

Funding acknowledgements

Swiss National Science Foundation:
nccr – on the move:

Open access

false

ISSN

0022-2836

Ausgabe der Veröffentlichung

3

Zeitschrift

Journal of molecular biology

Schlüsselwörter

Escherichia coli
Glutamic Acid
Arginine
Histidine
Ankyrins
Circular Dichroism
Combinatorial Chemistry Techniques
Reproducibility of Results
Protein Engineering
Temperature
Binding Sites
Amino Acid Sequence
Ankyrin Repeat
Consensus Sequence
Protein Conformation
Protein Structure, Secondary
Protein Structure, Tertiary
Protein Binding
Protein Denaturation
Point Mutation
Hydrogen Bonding
Hydrogen-Ion Concentration
Solubility
Light
Scattering, Radiation
Thermodynamics
Models, Chemical
Computer Simulation
Molecular Sequence Data
Hot Temperature
Hydrophobic and Hydrophilic Interactions

Sprache

eng

Medium

Print-Electronic

Online publication date

2007

Paginierung

837 - 854

Datum der Veröffentlichung

2008

Status

Published

Datum der Datenerfassung

2007

Titel

Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.

Sub types

Comparative Study
Research Support, Non-U.S. Gov't
Journal Article

Ausgabe der Zeitschrift

375

Datenquelle: Europe PubMed Central

Abstract

Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations. Notably, a stable intermediate is found in experimental equilibrium unfolding studies of one of the simulated consensus proteins. On the basis of simulation results, this intermediate is interpreted to represent a conformation with a denatured C-terminal repeat. To validate this interpretation, constructs without C-terminal capping repeat were prepared and did not show this intermediate in equilibrium unfolding experiments. Conversely, the capping repeats were found to be essential for efficient folding in the cell and for avoiding aggregation, presumably because of their highly charged surface. To design a capping repeat conferring similar solubility properties yet even higher stability, eight point mutations adapting the C-cap to the consensus AR and adding a three-residue extension at the C-terminus were predicted in silico and validated experimentally. The in vitro full-consensus proteins were also compared with a previously published designed AR protein, E3_5, whose internal repeats show 80% identity in primary sequence. A detailed analysis of the simulations suggests that networks of salt bridges between beta-hairpins, as well as additional interrepeat hydrogen bonds, contribute to the extraordinary stability of the full consensus.

Date of acceptance

2007

Autoren

Gianluca Interlandi
Svava K Wetzel
Giovanni Settanni
Andreas Plückthun
Amedeo Caflisch

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/18048057

DOI

10.1016/j.jmb.2007.09.042

eISSN

1089-8638

Ausgabe der Veröffentlichung

3

Zeitschrift

J Mol Biol

Schlüsselwörter

Amino Acid Sequence
Ankyrin Repeat
Ankyrins
Arginine
Binding Sites
Circular Dichroism
Combinatorial Chemistry Techniques
Computer Simulation
Consensus Sequence
Escherichia coli
Glutamic Acid
Histidine
Hot Temperature
Hydrogen Bonding
Hydrogen-Ion Concentration
Hydrophobic and Hydrophilic Interactions
Light
Models, Chemical
Molecular Sequence Data
Point Mutation
Protein Binding
Protein Conformation
Protein Denaturation
Protein Engineering
Protein Structure, Secondary
Protein Structure, Tertiary
Reproducibility of Results
Scattering, Radiation
Solubility
Temperature
Thermodynamics

Sprache

eng

Country

Netherlands

Paginierung

837 - 854

PII

S0022-2836(07)01222-3

Datum der Veröffentlichung

2008

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2008

Titel

Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.

Sub types

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

375

Datenquelle: PubMed

Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments

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