Selective ¹H^α NMR Methods Reveal Functionally Relevant Proline cis/trans Isomers in Intrinsically Disordered Proteins: Characterization of Minor Forms, Effects of Phosphorylation, and Occurrence in Proteome

Abstract

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins,a nd to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two 1 H a -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 %a mounts;m oreover,w es how the consequences of CK2 phosphorylation on the cis/trans-proline equilibrium. Using our results and available literature data we perform astatistical analysis on how the amino acid type effects the cis/trans-proline distribution. The methods are applicable under physiological conditions,they can contribute to find key proline isomers in proteins,and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes.

Probing Peptidylprolyl Bond cis/trans Status Using Distal 19F NMR Reporters

“…It was also clear that polar side chains, for example, Ser (12 %), Thr (13 %) Asp (13.5 %) promote a slightly higher cis ‐Pro population in X‐Pro systems compared with Ala‐Pro (10.5 %), while Arg (8 %) or Lys (9 %) in this position leads to a subtle decrease in the cis ‐Pro population. These results corroborate the observations from Sebak and co‐workers in their statistical analysis of cis ‐Pro populations in intrinsically disordered proteins [15] . Another interesting observation was the relatively high % cis for the Tyr‐Pro‐Gly (31 %) and Tyr‐Pro‐Pro (39 %) peptides, which are approximately equivalent to the Aro‐Pro‐Aro cis ‐populations.…”

“…These results corroborate the observations from Sebak and co-workers in their statistical analysis of cis-Pro populations in intrinsically disordered proteins. [15] Another interesting observation was the relatively high %cis for the Tyr-Pro-Gly (31 %) and Tyr-Pro-Pro (39 %) peptides, which are approximately equivalent to the Aro-Pro-Aro cis-populations.…”

“…NMR techniques provide structural information at the molecular level and can be useful in understanding the finer details of protein and peptide folding at specific sites, particularly with respect to proline isomerism. [14,15] Due to the relatively slow exchange rates between cis and trans conformers, 1 H NMR analysis of X-Pro systems typically affords two sets of signals, allowing the measurement of cis:trans. [9][10][11][12]16] However, this can create complexity in the analysis and in many cases, 2D NMR approaches are required to resolve specific resonances.…”

“…Most of the studies described have used NMR‐based approaches to detect and/or quantify prolyl conformers. NMR techniques provide structural information at the molecular level and can be useful in understanding the finer details of protein and peptide folding at specific sites, particularly with respect to proline isomerism [14,15] . Due to the relatively slow exchange rates between cis and trans conformers, 1 H NMR analysis of X‐Pro systems typically affords two sets of signals, allowing the measurement of cis : trans [9–12,16] .…”

The influence of random-coil chemical shifts on the assessment of structural propensities in folded proteins and IDPs

“…The p53TAD 1-60 construct has been studied in detail by our group earlier. 131,132 In the followings, from the available atom types, we limit ourselves to C a environments. Chemical shis of C a are generally considered one of, if not the, most sensitive parameters to secondary structure and structural propensities and have been preferred over their counterparts in various cases.…”