How many ionizable groups can sit on a protein hydrophobic core?
Article first published online: 30 SEP 2011
Copyright © 2011 Wiley Periodicals, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 80, Issue 1, pages 1–7, January 2012
How to Cite
Garcia-Seisdedos, H., Ibarra-Molero, B. and Sanchez-Ruiz, J. M. (2012), How many ionizable groups can sit on a protein hydrophobic core?. Proteins, 80: 1–7. doi: 10.1002/prot.23166
- Issue published online: 13 DEC 2011
- Article first published online: 30 SEP 2011
- Accepted manuscript online: 30 AUG 2011 10:49AM EST
- Manuscript Accepted: 5 AUG 2011
- Manuscript Revised: 29 JUL 2011
- Manuscript Received: 21 JAN 2011
- Spanish Ministry of Science and Innovation. Grant Numbers: BIO2009-09562, CSD2009-00088
- Junta de Andalucia. Grant Number: CVI-1668
- protein stability;
- mutation effects;
- disruptive mutations;
- protein robustness;
- polar group burial;
- enzyme catalysis
Full or partial burial of ionizable groups in the hydrophobic interior of proteins underlies the large modulation in group properties (modified pK value, high nucleophilicity, enhanced capability of interaction with chemical moieties of the substrate, etc.) linked to biological function. Indeed, the few internal ionizable residues found in proteins are known to play important functional roles in catalysis and, in general, in energy transduction processes. However, ionizable-group burial is expected to be seriously disruptive and, it is important to note, most functional sites contain not just one, but several ionizable residues. Hence, the adaptations involved in the development of function in proteins (through in vitro engineering or during the course of natural evolution) are not fully understood. Here, we explore experimentally how proteins respond to the accumulation of hydrophobic-to-ionizable residue substitutions. For this purpose, we have constructed a combinatorial library targeting a hydrophobic cluster in a consensus-engineered, stabilized form of a small model protein. Contrary to naïve expectation, half of the variants randomly selected from the library are soluble, folded, and active, despite including up to four mutations. Furthermore, for these variants, the dependence of stability with the number of mutations is not synergistic and catastrophic, but smooth and approximately linear. Clearly, stabilized protein scaffolds may be robust enough to withstand many disruptive hydrophobic-to-ionizable residue mutations, even when they are introduced in the same region of the structure. These results should be relevant for protein engineering and may have implications for the understanding of the early evolution of enzymes. Proteins 2012; © 2011 Wiley Periodicals, Inc.