Phosphorylation tunes elongation propensity and cohesiveness of INCENP's intrinsically disordered region.

The inner centromere protein, INCENP, is crucial for correct chromosome segregation during mitosis. It connects the kinase Aurora B to the inner centromere allowing this kinase to dynamically access its kinetochore targets. However, the function of its central, 440-residue long intrinsically disordered region (IDR) and its multiple phosphorylation sites is unclear. Here, we determined the conformational ensemble of INCENP's IDR, systematically varying the level of phosphorylation, using all-atom and coarse-grain molecular dynamics simulations. Our simulations show that phosphorylation expands INCENP's IDR, both locally and globally, mainly by increasing its overall net charge. The disordered region undergoes critical globule-to-coil conformational transitions and the transition temperature non-monotonically depends on the degree of phosphorylation, with a mildly phosphorylated case of neutral net charge featuring the highest collapse propensity. The IDR transitions from a multitude of globular states, accompanied by several specific internal contacts that reduce INCENP length by loop formation, to weakly interacting and highly extended coiled conformations. Phosphorylation critically shifts the population between these two regimes. It thereby influences cohesiveness and phase behavior of INCENP IDR assemblies, a feature presumably relevant for INCENP's function in the chromosomal passenger complex. Overall, we propose the disordered region of INCENP to act as a phosphorylation-regulated and length-variable component, within the previously defined "dog-leash" model, that thereby regulates how Aurora B reaches its targets for proper chromosome segregation.

Relative Quantitation of Beta-Amyloid Peptide Isomers with Simultaneous Isomerization of Multiple Aspartic Acid Residues by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry can be used for rapid quantitation of peptides with various post-translational modifications (PTM), even if they do not shift the mass of the native peptide. Previously, it was shown that MALDI-TOF MS can be used for quantitation of isoD7 beta-amyloid 1-42 peptide. On the basis of the differences in the collision-induced dissociation fragmentation pattern of native Aß, isoD7 Aß, isoD23 Aß, and isoD7_23 peptide (a di-isomerized peptide with both isomerization of D7 and D23 residues), we developed a MALDI-TOF-based method for simultaneous quantitation of all of these isoforms. Using multivariate regression for analysis of fragment MS data, the method allows the determination of the molar fractions of all of these isoforms with up to 16% error for mixtures with 2 pmol total amount of the beta-amyloid peptide.

Probabilistic model applied to ion abundances in product-ion spectra: quantitative analysis of aspartic acid isomerization in peptides.

Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization. Graphical Abstract.