2GJF

NMR structure of the computationally designed procarboxypeptidase-A (1AYE) domain

Summary for 2GJF

• Entry DOI: 10.2210/pdb2gjf/pdb
• Related: 1VJQ
• NMR Information: BMRB: 7102
• Descriptor: DESIGNED PROTEIN (1 entity in total)
• Functional Keywords: designed protein, procarboxypeptidase, de novo protein
• Total number of polymer chains: 2
• Total formula weight: 18022.41

Authors

Reichow, S. (deposition date: 2006-03-30, release date: 2007-01-16, Last modification date: 2024-05-29)

Primary citation

Dantas, G.,Corrent, C.,Reichow, S.L.,Havranek, J.J.,Eletr, Z.M.,Isern, N.G.,Kuhlman, B.,Varani, G.,Merritt, E.A.,Baker, D.
High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design.
J.Mol.Biol., 366:1209-1221, 2007

PubMed Abstract: Recent efforts to design de novo or redesign 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 have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.

PubMed: 17196978
DOI: 10.1016/j.jmb.2006.11.080

Experimental method

SOLUTION NMR