Pathways to folding, nucleation events and native geometry

We perform extensive Monte Carlo simulations of a lattice model and the Go potential to investigate the existence of folding pathways at the level of contact cluster formation for two native structures with markedly different geometries. Our analysis of folding pathways revealed a common underlying folding mechanism, based on nucleation phenomena, for both protein models. However, folding to the more complex geometry (i.e. that with more non-local contacts) is driven by a folding nucleus whose geometric traits more closely resemble those of the native fold. For this geometry folding is clearly a more cooperative process.Comment: Accepted in J. Chem. Phy

Steady-state fluctuations of a genetic feedback loop:an exact solution

Genetic feedback loops in cells break detailed balance and involve bimolecular reactions; hence exact solutions revealing the nature of the stochastic fluctuations in these loops are lacking. We here consider the master equation for a gene regulatory feedback loop: a gene produces protein which then binds to the promoter of the same gene and regulates its expression. The protein degrades in its free and bound forms. This network breaks detailed balance and involves a single bimolecular reaction step. We provide an exact solution of the steady-state master equation for arbitrary values of the parameters, and present simplified solutions for a number of special cases. The full parametric dependence of the analytical non-equilibrium steady-state probability distribution is verified by direct numerical solution of the master equations. For the case where the degradation rate of bound and free protein is the same, our solution is at variance with a previous claim of an exact solution (Hornos et al, Phys. Rev. E {\bf 72}, 051907 (2005) and subsequent studies). We show explicitly that this is due to an unphysical formulation of the underlying master equation in those studies.Comment: 31 pages, 3 figures. Accepted for publication in the Journal of Chemical Physics (2012

Protein folding rates correlate with heterogeneity of folding mechanism

By observing trends in the folding kinetics of experimental 2-state proteins at their transition midpoints, and by observing trends in the barrier heights of numerous simulations of coarse grained, C-alpha model, Go proteins, we show that folding rates correlate with the degree of heterogeneity in the formation of native contacts. Statistically significant correlations are observed between folding rates and measures of heterogeneity inherent in the native topology, as well as between rates and the variance in the distribution of either experimentally measured or simulated phi-values.Comment: 11 pages, 3 figures, 1 tabl

The checkpoint role of Cdc18 in fission yeast.

The highly conserved eukaryotic checkpoints keep tight control over cell cycle progression, arresting the cell in response to incomplete DNA replication or DNA damage. In fission yeast, Rad3 (functional homologue of ATM, mutated in ataxia telangiectasia, and structural homologue of ATR, ataxia telangiectasia and rad3 related) is necessary for activation of both replication and damage checkpoints. However, despite the identification of many checkpoint genes, the actual sequence of upstream events leading to Rad3 activation remains unclear. The aim of my project was to identify and characterise the Rad3- dependent DNA damage/perturbed replication sensors and checkpoint activators. A genetic screen was carried out in fission yeast, using the working hypothesis that overexpression of these sensors/checkpoint activators would ectopically induce a Rad3-dependent block over mitosis in the absence of DNA damage or disturbed replication. The screen identified several genes of which the DNA replication initiation factor Cdc18/CDC6 had the strongest and most reproducible phenotype. Cdc18 is essential to prevent mitosis during S phase. I chose to concentrate on characterisation of the Rad3-dependent checkpoint role of Cdc18. The actual level of Cdc18 is important for producing the Rad3-dependent cell cycle block. A stabilised Cdc18 protein, mutated at the conserved CDK (cyclin dependent kinase) consensus sites also caused a Rad3-dependent cell cycle arrest, and was more stable and easier to manipulate than the screen- derived clone. Genetic crosses demonstrated Cdc18 acts early on in the checkpoint pathway, and through Crb2/Chk1. There was no gross DNA damage or detectable replication intermediates in the presence of elevated or stabilised Cdc18 levels. I also found that artificial depletion of Cdc18 during an S phase block results in loss of the checkpoint but not the replication structures, uncoupling the maintenance of replication forks from the maintenance of the mitotic block. An unexpected consequence of Cdc18 stabilisation was an increase in the size and variability of chromosome III on pulsed field gel electrophoresis. This localised to an expansion of Sfi1 restriction fragments containing the rDNA repeats. In conclusion, Cdc18 stabilisation activates a Rad3-dependent checkpoint in the absence of apparent re-replication, which is associated with an expansion of the rDNA repeats on chromosome III. Two models are proposed. In the first, Cdc18 induces low level genome wide replication, that is undetectable but sufficient for checkpoint activation. This leads to increased recombination with unequal crossover events in the rDNA repeats on chromosome III, with subsequent repeat expansion. In the second, the increased levels of Cdc18 directly activate the cell cycle checkpoint independently of the concurrent expansion of chromosome III

Limitations of the stochastic quasi-steady-state approximation in open biochemical reaction networks

The application of the quasi-steady-state approximation to the Michaelis-Menten reaction embedded in large open chemical reaction networks is a popular model reduction technique in deterministic and stochastic simulations of biochemical reactions inside cells. It is frequently assumed that the predictions of the reduced master equations obtained using the stochastic quasi-steady-state approach are in very good agreement with the predictions of the full master equations, provided the conditions for the validity of the deterministic quasi-steady-state approximation are fulfilled. We here use the linear-noise approximation to show that this assumption is not generally justified for the Michaelis-Menten reaction with substrate input, the simplest example of an open embedded enzyme reaction. The reduced master equation approach is found to considerably overestimate the size of intrinsic noise at low copy numbers of molecules. A simple formula is obtained for the relative error between the predictions of the reduced and full master equations for the variance of the substrate concentration fluctuations. The maximum error is reached when modeling moderately or highly efficient enzymes, in which case the error is approximately 30%. The theoretical predictions are validated by stochastic simulations using experimental parameter values for enzymes involved in proteolysis, gluconeogenesis and fermentation.Comment: Main text (5 pages, 1 figure) and Supplementary information (2 pages), accepted by J. Chem. Phys. (Communication

Clinical, Imaging and Neurogenetic Features of Patients with Gliomatosis Cerebri Referred to a Tertiary Neuro-Oncology Centre

Introduction: Gliomatosis cerebri describes a rare growth pattern of diffusely infiltrating glioma. The treatment options are limited and clinical outcomes remain poor. To characterise this population of patients, we examined referrals to a specialist brain tumour centre. Methods: We analysed demographic data, presenting symptoms, imaging, histology and genetics, and survival in individuals referred to a multidisciplinary team meeting over a 10-year period. Results: In total, 29 patients fulfilled the inclusion criteria with a median age of 64 years. The most common presenting symptoms were neuropsychiatric (31%), seizure (24%) or headache (21%). Of 20 patients with molecular data, 15 had IDH wild-type glioblastoma, with an IDH1 mutation most common in the remainder (5/20). The median length of survival from MDT referral to death was 48 weeks (IQR 23 to 70 weeks). Contrast enhancement patterns varied between and within tumours. In eight patients who had DSC perfusion studies, five (63%) had a measurable region of increased tumour perfusion with rCBV values ranging from 2.8 to 5.7. A minority of patients underwent MR spectroscopy with 2/3 (66.6%) false-negative results. Conclusions: Gliomatosis imaging, histological and genetic findings are heterogeneous. Advanced imaging, including MR perfusion, could identify biopsy targets. Negative MR spectroscopy does not exclude the diagnosis of glioma

Genomic Prognosticators and Extent of Resection in Molecularly Subtyped World Health Organization Grade II and III Gliomas–A Single-Institution, Nine-Year Data

BACKGROUND: WHO grade II and III IDH wild-type (IDH-wt) gliomas are often treated as WHO grade IV glioblastomas. However, cumulative evidence indicates that IDH mutation status alone is insufficient in predicting survival. The current study examines molecular and clinical markers to further prognostically stratify WHO grade II and III gliomas, in particular, IDH-wt. METHODS: A single institution's records were retrospectively reviewed for molecularly stratified WHO grade II and grade III gliomas over a nine-year period (2010-2019). Clinical data, IDH1/IDH2 status, EGFR amplification and other molecular markers were recorded and correlated to the study outcomes. These were defined as progression-free survival (PFS), overall survival (OS) and time to malignant progression (TtMP). RESULTS: 167 and 42 WHO grade II and III gliomas, respectively, were identified, totalling 209 cases with 157 IDH1/2 mutated and 52 IDH wild-type tumours. The presence of IDH1/2 mutation was associated with longer OS (p<0.0001) and PFS (p<0.0001) but not with TtMP (p=0.314). Lack of EGFR amplification, younger age, greater extent of resection (EOR) (≥80%) were identified as independent, favourable prognostic factors. In the IDH-wt cohort, multivariate analysis indicated that older age (p=0.003) and lesser EOR (<80%) (p=0.007) are associated with worse OS. Additionally, EGFR amplification showed a trend toward shorter OS in the IDH-wt cohort (p=0.073). CONCLUSIONS: IDH1/2 mutation favours longer OS and PFS but does not protect from malignant progression. Lack of EGFR amplification, older age and greater EOR are favourable OS prognosticators. In the IDH-wt cohort, older age and lesser EOR were linked to worse OS

Dynamical chaos and power spectra in toy models of heteropolymers and proteins

The dynamical chaos in Lennard-Jones toy models of heteropolymers is studied by molecular dynamics simulations. It is shown that two nearby trajectories quickly diverge from each other if the heteropolymer corresponds to a random sequence. For good folders, on the other hand, two nearby trajectories may initially move apart but eventually they come together. Thus good folders are intrinsically non-chaotic. A choice of a distance of the initial conformation from the native state affects the way in which a separation between the twin trajectories behaves in time. This observation allows one to determine the size of a folding funnel in good folders. We study the energy landscapes of the toy models by determining the power spectra and fractal characteristics of the dependence of the potential energy on time. For good folders, folding and unfolding trajectories have distinctly different correlated behaviors at low frequencies.Comment: 8 pages, 9 EPS figures, Phys. Rev. E (in press