Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

Departamento de Química Física Facultad de Ciencias Universidad de Granada Av Fuentenueva s n 18071 Granada Spain

Departamento de Química Física Unidad de Excelencia de Química aplicada a Biomedicina y Medioambiente e Instituto de Biotecnología Facultad de Ciencias Universidad de Granada Av Fuentenueva s n 18071 Granada Spain

Department of Biotechnology Bhupat and Jyoti Mehta School of Biosciences Indian Institute of Technology Madras Chennai 600036 India

Institute of Biotechnology of the Czech Academy of Sciences BioCeV Prumyslova 595 252 50 Vestec Czech Republic

Institute of Microbiology of the Czech Academy of Sciences BioCeV Prumyslova 595 252 50 Vestec Czech Republic

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