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Title: High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design

Abstract

Recent efforts to redesign or de novo design the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol, RosettaDesign, to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal / mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we design and characterize a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations – one mutant gains over 5 kcal/mol of stability over the wild-type protein with only four amino-acid changes. We explore the relationship between force-field resolution and conformational sampling by comparing the experimentally determined free energies of themore » overall design and these focused subsets of mutations to those predicted using force fields of different resolution and both fixed and flexible backbone sampling protocols.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
902664
Report Number(s):
PNNL-SA-51259
Journal ID: ISSN 0022-2836; JMOBAK; 16722a; KP1704020; TRN: US200717%%604
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Molecular Biology, 366(4):1209-1221; Journal Volume: 366; Journal Issue: 4
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ACCURACY; CRYSTAL STRUCTURE; DESIGN; MUTANTS; MUTATIONS; PROTEINS; RESOLUTION; SAMPLING; STABILITY; STABILIZATION; THERMODYNAMICS; Environmental Molecular Sciences Laboratory

Citation Formats

Dantas, Gautam, Corrent, Colin, Reichow, Steve L., Havranek, James J., Eletr, Ziad, Isern, Nancy G., Kuhlman, Brian, Varani, Gabriele, Merritt, Ethan, and Baker, David. High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design. United States: N. p., 2007. Web. doi:10.1016/j.jmb.2006.11.080.
Dantas, Gautam, Corrent, Colin, Reichow, Steve L., Havranek, James J., Eletr, Ziad, Isern, Nancy G., Kuhlman, Brian, Varani, Gabriele, Merritt, Ethan, & Baker, David. High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design. United States. doi:10.1016/j.jmb.2006.11.080.
Dantas, Gautam, Corrent, Colin, Reichow, Steve L., Havranek, James J., Eletr, Ziad, Isern, Nancy G., Kuhlman, Brian, Varani, Gabriele, Merritt, Ethan, and Baker, David. Fri . "High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design". United States. doi:10.1016/j.jmb.2006.11.080.
@article{osti_902664,
title = {High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design},
author = {Dantas, Gautam and Corrent, Colin and Reichow, Steve L. and Havranek, James J. and Eletr, Ziad and Isern, Nancy G. and Kuhlman, Brian and Varani, Gabriele and Merritt, Ethan and Baker, David},
abstractNote = {Recent efforts to redesign or de novo design the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol, RosettaDesign, to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal / mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we design and characterize a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations – one mutant gains over 5 kcal/mol of stability over the wild-type protein with only four amino-acid changes. We explore the relationship between force-field resolution and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using force fields of different resolution and both fixed and flexible backbone sampling protocols.},
doi = {10.1016/j.jmb.2006.11.080},
journal = {Journal of Molecular Biology, 366(4):1209-1221},
number = 4,
volume = 366,
place = {United States},
year = {Fri Mar 02 00:00:00 EST 2007},
month = {Fri Mar 02 00:00:00 EST 2007}
}