Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins

Autoren

Svava K Wetzel
Giovanni Settanni
Manca Kenig
H Kaspar Binz
Andreas Plückthun

DOI

10.1016/j.jmb.2007.11.046

ISSN

0022-2836

Ausgabe der Veröffentlichung

1

Zeitschrift

Journal of Molecular Biology

Sprache

en

Paginierung

241 - 257

Datum der Veröffentlichung

2008

Status

Published

Herausgeber

Elsevier BV

Herausgeber URL

http://dx.doi.org/10.1016/j.jmb.2007.11.046

Datum der Datenerfassung

2019

Titel

Folding and Unfolding Mechanism of Highly Stable Full-Consensus Ankyrin Repeat Proteins

Ausgabe der Zeitschrift

376

Datenquelle: Crossref

Abstract

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins ( approximately 400-800 s(-1)), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10(4) with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.

Addresses

Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Autoren

Svava K Wetzel
Giovanni Settanni
Manca Kenig
H Kaspar Binz
Andreas Plückthun

DOI

10.1016/j.jmb.2007.11.046

eISSN

1089-8638

Externe Identifier

PubMed Identifier: 18164721

Open access

false

ISSN

0022-2836

Ausgabe der Veröffentlichung

1

Zeitschrift

Journal of molecular biology

Schlüsselwörter

Escherichia coli
Ankyrins
Spectrometry, Fluorescence
Circular Dichroism
Gene Expression
Amino Acid Sequence
Ankyrin Repeat
Protein Folding
Kinetics
Models, Molecular
Molecular Sequence Data

Sprache

eng

Medium

Print-Electronic

Online publication date

2007

Paginierung

241 - 257

Datum der Veröffentlichung

2008

Status

Published

Datum der Datenerfassung

2008

Titel

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins.

Sub types

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

Ausgabe der Zeitschrift

376

Datenquelle: Europe PubMed Central

Abstract

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins ( approximately 400-800 s(-1)), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10(4) with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.

Date of acceptance

2007

Autoren

Svava K Wetzel
Giovanni Settanni
Manca Kenig
H Kaspar Binz
Andreas Plückthun

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/18164721

DOI

10.1016/j.jmb.2007.11.046

eISSN

1089-8638

Ausgabe der Veröffentlichung

1

Zeitschrift

J Mol Biol

Schlüsselwörter

Amino Acid Sequence
Ankyrin Repeat
Ankyrins
Circular Dichroism
Escherichia coli
Gene Expression
Kinetics
Models, Molecular
Molecular Sequence Data
Protein Folding
Spectrometry, Fluorescence

Sprache

eng

Country

Netherlands

Paginierung

241 - 257

PII

S0022-2836(07)01530-6

Datum der Veröffentlichung

2008

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2008

Titel

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins.

Sub types

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

Ausgabe der Zeitschrift

376

Datenquelle: PubMed

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins

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