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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}
}
  • Achieving atomic-level resolution in the computational design of a protein structure remains a challenging problem despite recent progress. Rigorous experimental tests are needed to improve protein design algorithms, yet studies of the structure and dynamics of computationally designed proteins are very few. The NMR structure and backbone dynamics of a redesigned protein of 96 amino acids are compared here with the design target, human U1A protein. We demonstrate that the redesigned protein reproduces the target structure to within the uncertainty of the NMR coordinates, even as 65 out of 96 amino acids were simultaneously changed by purely computational methods. Themore » dynamics of the backbone of the redesigned protein also mirror those of human U1A, suggesting that the protein design algorithm captures the shape of the potential energy landscape in addition to the local energy minimum.« less
  • This present study analyzed the correlation between nuclear morphology and the protein synthesis rate. The latter was assayed by measuring the /sup 14/C-1-leucine incorporation rate utilizing the technique of quantitative /sup 14/C-autoradiography. The labeled cells were first classified according to conventional cytological criteria into four groups of increasing maturity. Following grain counting the nuclei were Feulgen-stained, and after removal of the silver grains, the local nuclear optical densities were evaluated by scanning. There was a nonlinear relationship between the protein synthesis rate and features representing the degree of chromatin condensation. This nonlinearity was explained by the mediator function of RNA,more » predominantly mRNA. The amount of protein produced at a given time depends on the transcriptional activity of the chromatin, the frequency of mitotic divisions partitioning the mRNA, and the half-life of the mRNA. It was concluded that the chromatin texture of erythroblasts reflects three different metabolic activities: the rate of DNA strand duplication, the transcriptional activity for structural proteins enabling a cell to grow and cycle, and for functional proteins, particularly hemoglobin. Since these three activities appear to be synchronized, it is understandable that functional as well as textural features can be used to perform a supervised classification by means of multivariate analysis. Application of the five most significant features allowed a consistent classification of the erythroblasts into the various cytological compartments in 76.7% of all cases, while the classification error of the observer amounted to 13.7%.« less
  • In order to understand the detailed association of the macro-molecules of the structure of the protein, a high resolution structural analysis was performed. Large, single layered arrays of the surface layer protein have been obtained for this purpose by means of extensive heating in high CaCl/sub 2/. The computer processed image reveals a pore of about 10 Angstrom diameter at the 6-fold symmetry center; the handedness of the images is quite evident. The individual molecular envelope of the protein monomers are apparent and details of the protein-protein contact at the three-fold lattice positions emerge.
  • Human replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, homologous recombination, and excision repair. The authors have previously reported the cloning and bacterial overexpression of the three RPA genes and have mapped them to chromosome 1 (RPA2), chromosome 7 (RPA3), and chromosome 17 (RP1). They have now obtained yeast strains with artificial chromosomes carrying the three human RPA genes and report the more detailed genomic mapping of RPA. RPA1 was mapped to chromosome 17p13.3 using a combination of PCR amplification of somatic cell hybrids and radiation hybrids containing chromosome 17 fragments.more » RPA2 was mapped to chromosome 1p35 by PCR amplification of somatic cell hybrids of chromosome 1 and by fluorescence in situ hybridization. RPA3 was mapped to chromosome 7p22 by Southern analysis and PCR amplification of somatic cell hybrids of chromosome 7 as well as fluorescence in situ hybridization. Since RPA is an essential component of major metabolic events affecting DNA, the physical mapping of the genes for it may help elucidate the biochemical basis of genetic disorders involving DNA metabolism. 73 refs., 8 figs., 1 tab.« less