Fluorescence spectroscopy of H‐ras transfected murine fibroblasts: A comparison with Monte Carlo simulations

Autofluorescence properties of tissues have been widely used to diagnose various types of malignancies. In this study, we measured the autofluorescence properties of H‐ras transfected murine fibroblasts and the counterpart control cells. The pair of cells is genetically identical except for the transfected H‐ras gene. We applied Monte Carlo simulations to evaluate the relative contributions of Rayleigh and Mie scattering effects towards fluorescence in an in vitro model system of normal and H‐ras transfected fibroblasts. The experimental results showed that fluorescence emission intensity was higher for normal cells than the malignant counterpart cells by about 30%. In normal cells, linearity in emission intensity was observed for cell densities of up to 1.0 × 10⁶ cells/ml whereas for transformed cells it was up to 1.4 × 10⁶ cells/ml. Nuclear volume changes give good account for the differences in the intrinsic fluorescence between normal and malignant cells. The Monte Carlo (MC) code, newly developed for this study, explains both predominant experimental features: the large fluorescence intensity differences between the transfected and the corresponding control cells as well as the phenomena of the red shift in the excitation spectra as a function of cell density. The contribution of Rayleigh scattering was found to be predominant compared to Mie scattering. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 132–140, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Coarse‐grained modeling of the intrinsically disordered protein Histatin 5 in solution: Monte Carlo simulations in combination with SAXS

Monte Carlo simulations and coarse‐grained modeling have been used to analyze Histatin 5, an unstructured short cationic salivary peptide known to have anticandidical properties. The calculated scattering functions have been compared with intensity curves and the distance distribution function P(r) obtained from small angle X‐ray scattering (SAXS), at both high and low salt concentrations. The aim was to achieve a molecular understanding and a physico‐chemical insight of the obtained SAXS results and to gain information of the conformational changes of Histatin 5 due to altering salt content, charge distribution, and net charge. From a modeling perspective, the accuracy of the electrostatic interactions are of special interest. The used coarse‐grained model was based on the primitive model in which charged hard spheres differing in charge and in size represent the ionic particles, and the solvent only enters the model through its relative permittivity. The Hamiltonian of the model comprises three different contributions: (i) excluded volumes, (ii) electrostatic, and (iii) van der Waals interactions. Even though the model can be considered as gross omitting all atomistic details, a great correspondence is obtained with the experimental results. Proteins 2016; 84:777–791. © 2016 Wiley Periodicals, Inc.     

Protein conformational transitions coupled to binding in molecular recognition of unstructured proteins: hierarchy of structural loss from all-atom Monte Carlo simulations of p27Kip1 unfolding-unbinding and structural determinants of the binding mechanism

Conformational transitions coupled to binding are studied for the p27(Kip1) protein which undergoes a functional disorder-to-order folding transition during tertiary complex formation with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) binary complex. Temperature-induced Monte Carlo simulations of p27(Kip1) unfolding-unbinding carried out from the crystal structure of the tertiary complex have revealed a systematic trend in the hierarchy of structural loss for p27(Kip1) and a considerable difference in mobility of p27(Kip1) secondary structure elements. The most persistent interactions of p27(Kip1) at the intermolecular interface during unfolding-unbinding simulations are formed by beta-hairpin and beta-strand that on average maintain their structural integrity considerably longer than other p27(Kip1) elements. We have found that the ensemble of unfolded p27(Kip1) conformations is characterized by transitions between mostly unbound, collapsed conformations and entropically favorable p27(Kip1) conformations, which are weakly bound to the cyclin A side of the binary complex. The results of this study are consistent with the experimental evidence pointing to this region of the intermolecular interface as a potential initiation docking site during binding reaction and may reconcile conflicting experimental hypotheses on the recognition of substrate recruitment motifs.