High-resolution Structural and Thermodynamic Analysis of Extreme Stabilization of Human Procarboxypeptidase by Computational Protein Design
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.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 902664
- Report Number(s):
- PNNL-SA-51259; JMOBAK; 16722a; KP1704020; TRN: US200717%%604
- Journal Information:
- Journal of Molecular Biology, 366(4):1209-1221, Vol. 366, Issue 4; ISSN 0022-2836
- Country of Publication:
- United States
- Language:
- English
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