Selective 1Hα NMR methods to reveal functionally relevant proline cis/trans isomers in IDPs: characterization of minor forms, effects of phosphorylation and occurrence in proteome.

Identification of proline cis/trans isomers appearing in several regulatory mechanisms of proteins and characterization of minor species present due to the conformational heterogeneity in IDPs is highly important. To obtain residue level information on these mobile systems we introduce two 1 H α -detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4-15% amounts, moreover we show the consequences of CK2 phosphorylation on the cis/trans -proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans proline distribution. The methods are applicable under physiological conditions, and they can contribute to find key proline isomers in proteins and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes.