Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain

Ben T Porebski, Adrian Nickson, David E Hoke, Morag R Hunter, Liguang Zhu, Sheena McGowan, Geoffrey I Webb, Ashley M Buckle

Research output: Contribution to journalArticleResearchpeer-review

17 Citations (Scopus)

Abstract

Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100 degrees C, a DeltaGD-N of 15.5 kcal mol(-1) and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 A and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.
Original languageEnglish
Pages (from-to)67-78
Number of pages12
JournalProtein Engineering Design and Selection
Volume28
Issue number3
DOIs
Publication statusPublished - 2015

Keywords

  • Consensus design
  • Fibronectin type III
  • FN3
  • Molecular dynamics
  • Stability

Cite this

@article{91738de1635e40d19d54c35d772db8d4,
title = "Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain",
abstract = "Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100 degrees C, a DeltaGD-N of 15.5 kcal mol(-1) and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 A and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.",
keywords = "Consensus design, Fibronectin type III, FN3, Molecular dynamics, Stability",
author = "Porebski, {Ben T} and Adrian Nickson and Hoke, {David E} and Hunter, {Morag R} and Liguang Zhu and Sheena McGowan and Webb, {Geoffrey I} and Buckle, {Ashley M}",
year = "2015",
doi = "10.1093/protein/gzv002",
language = "English",
volume = "28",
pages = "67--78",
journal = "Protein Engineering Design and Selection",
issn = "1741-0126",
publisher = "Oxford University Press",
number = "3",

}

Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain. / Porebski, Ben T; Nickson, Adrian; Hoke, David E; Hunter, Morag R; Zhu, Liguang; McGowan, Sheena; Webb, Geoffrey I; Buckle, Ashley M.

In: Protein Engineering Design and Selection, Vol. 28, No. 3, 2015, p. 67-78.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain

AU - Porebski, Ben T

AU - Nickson, Adrian

AU - Hoke, David E

AU - Hunter, Morag R

AU - Zhu, Liguang

AU - McGowan, Sheena

AU - Webb, Geoffrey I

AU - Buckle, Ashley M

PY - 2015

Y1 - 2015

N2 - Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100 degrees C, a DeltaGD-N of 15.5 kcal mol(-1) and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 A and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.

AB - Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100 degrees C, a DeltaGD-N of 15.5 kcal mol(-1) and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 A and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.

KW - Consensus design

KW - Fibronectin type III

KW - FN3

KW - Molecular dynamics

KW - Stability

UR - http://www.scopus.com/inward/record.url?scp=84983126623&partnerID=8YFLogxK

U2 - 10.1093/protein/gzv002

DO - 10.1093/protein/gzv002

M3 - Article

C2 - 25691761

VL - 28

SP - 67

EP - 78

JO - Protein Engineering Design and Selection

JF - Protein Engineering Design and Selection

SN - 1741-0126

IS - 3

ER -