Selective 1Hα NMR methods to reveal functionally relevant proline cis/trans isomers in IDPs

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and 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 H-1(alpha)-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, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes

Novel Lysine-Rich Delivery Peptides of Plant Origin ERD and Human S100: The Effect of Carboxyfluorescein Conjugation, Influence of Aromatic and Proline Residues, Cellular Internalization, and Penetration Ability

The need for novel drug delivery peptides is an important issue of the modern pharmaceutical research. Here, we test K-rich peptides from plant dehydrin ERD14 (ERD-A, ERD-B, and ERD-C) and the C-terminal CPP-resembling region of S100A4 (S100) using the 5(6)-carboxyfluorescein (Cf) tag at the N-terminus. Via a combined pH-dependent NMR and fluorescence study, we analyze the effect of the Cf conjugation/modification on the structural behavior, separately investigating the (5)-Cf and (6)-Cf forms. Flow cytometry results show that all peptides internalize; however, there is a slight difference between the cellular internalization of (5)- and (6)-Cf-peptides. We indicate the possible importance of residues with an aromatic sidechain and proline. We prove that ERD-A localizes mostly in the cytosol, ERD-B and S100 have partial colocalization with lysosomal staining, and ERD-C mainly localizes within vesicle-like compartments, while the uptake mechanism mainly occurs through energy-dependent paths

Assignment of the disordered, proline-rich N-terminal domain of the tumour suppressor p53 protein using 1HN and 1Hα-detected NMR measurements

Protein p53 is mostly known for playing a key role in tumour suppression, and mutations in the p53 gene are amongst the most frequent genomic events accompanying oncogenic transformation. Continuous research is conducted to target disordered proteins/protein regions for cancer therapy, for which atomic level information is also necessary. The disordered N-terminal part of p53 contains the transactivation and the proline-rich domains—which besides being abundant in proline residues—contains repetitive Pro-Ala motifs. NMR assignment of such repetitive, proline-rich regions is challenging due to the lack of amide protons in the 1 H N -detected approaches, as well as due to the small chemical shift dispersion. In the present study we perform the full assignment of the p53 1–100 region by applying a combination of 1 H N - and 1 H α -detected NMR experiments. We also show the increased information content when using real-time homo- and heteronuclear decoupled acquisition schemes. On the other hand, we highlight the presence of minor proline species, and using Pro-selective experiments we determine the corresponding cis or trans conformation. Secondary chemical shifts for (C α –C β ) atoms indicate the disordered nature of this region, with expected helical tendency for the TAD1 region. As the role of the proline-rich domain is yet not well understood our results can contribute to further successful investigations

Proline cis/trans Isomerization in Intrinsically Disordered Proteins and Peptides

Background: Intrinsically disordered proteins and protein regions (IDPs/IDRs) are important in diverse biological processes. Lacking a stable secondary structure, they display an ensemble of conformations. One factor contributing to this conformational heterogeneity is the proline cis/trans isomerization. The knowledge and value of a given cis/trans proline ratio are paramount, as the different conformational states can be responsible for different biological functions. Nuclear Magnetic Resonance (NMR) spectroscopy is the only method to characterize the two co-existing isomers on an atomic level, and only a few works report on these data. Methods: After collecting the available experimental literature findings, we conducted a statistical analysis regarding the influence of the neighboring amino acid types (i ± 4 regions) on forming a cis-Pro isomer. Based on this, several regularities were formulated. NMR spectroscopy was then used to define the cis-Pro content on model peptides and desired point mutations. Results: Analysis of NMR spectra prove the dependence of the cis-Pro content on the type of the neighboring amino acid—with special attention on aromatic and positively charged sidechains. Conclusions: Our results may benefit the design of protein regions with a given cis-Pro content, and contribute to a better understanding of the roles and functions of IDPs

The Disordered EZH2 Loop: Atomic Level Characterization by ¹H^N- and ¹H^α-Detected NMR Approaches, Interaction with the Long Noncoding HOTAIR RNA

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the 1HN-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, 1Hα-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407–Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR140 (1–140 nt) highlight that the Thr367–Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein–RNA interaction as a “fuzzy” complex

Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model

Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model Reversible serine proteinase inhibitors comprise 18 unrelated families. Each family has a distinct representative structure but contains a surface loop that adopts the same, canonical conformation in the enzyme–inhibitor complex. The Laskowski mechanism universally applies for the action of all canonical inhibitors independent of their scaffold, but it has two nontrivial extrapolations. Intrascaffolding additivity states that all enzyme-contacting loop residues act independently of each other, while interscaffolding additivity claims that these residues act independently of the scaffold. These theories have great importance for engineering proteinase inhibitors but have not been comprehensively challenged. Therefore, we tested the interscaffolding additivity theory by hard-randomizing all enzyme-contacting canonical loop positions of a Kazal- and a Pacifastin-scaffold inhibitor, displaying the variants on M13 phage, and selecting the libraries on trypsin and chymotrypsin. Directed evolution delivered different patterns on both scaffolds against both enzymes, which contradicts interscaffolding additivity. To quantitatively assess the extent of non-additivity, we measured the affinities of the optimal binding loop variants and their binding loop-swapped versions. While optimal variants have picomolar affinities, swapping the evolved loops results in up to 200,000-fold affinity loss. To decipher the underlying causes, we characterized the stability, overall structure and dynamics of the inhibitors with differential scanning calorimetry, circular dichroism and NMR spectroscopy and molecular dynamic simulations. These studies revealed that the foreign loop destabilizes the lower-stability Pacifastin scaffold, while the higher-stability Kazal scaffold distorts the foreign loop. Our findings disprove interscaffolding additivity and show that loop and scaffold form one integrated unit that needs to be coevolved to provide high-affinity inhibition