Automation of peak-tracking analysis of stepwise perturbed NMR spectra

6noWe describe a new algorithmic approach able to automatically pick and track the NMR resonances of a large number of 2D NMR spectra acquired during a stepwise variation of a physical parameter. The method has been named Trace in Track (TinT), referring to the idea that a gaussian decomposition traces peaks within the tracks recognised through 3D mathematical morphology. It is capable of determining the evolution of the chemical shifts, intensity and linewidths of each tracked peak.The performances obtained in term of track reconstruction and correct assignment on realistic synthetic spectra were high above 90% when a noise level similar to that of experimental data were considered. TinT was applied successfully to several protein systems during a temperature ramp in isotope exchange experiments. A comparison with a state-of-the-art algorithm showed promising results for great numbers of spectra and low signal to noise ratios, when the graduality of the perturbation is appropriate. TinT can be applied to different kinds of high throughput chemical shift mapping experiments, with quasi-continuous variations, in which a quantitative automated recognition is crucial.openopenBanelli, Tommaso; Vuano, Marco; Fogolari, Federico; Fusiello, Andrea; Esposito, Gennaro; Corazza, AlessandraBanelli, Tommaso; Vuano, Marco; Fogolari, Federico; Fusiello, Andrea; Esposito, Gennaro; Corazza, Alessandr

Probing the influence of citrate-capped gold nanoparticles on an amyloidogenic protein

Nanoparticles (NPs) are known to exhibit distinct physical and chemical properties compared with the same materials in bulk form. NPs have been repeatedly reported to interact with proteins, and this interaction can be exploited to affect processes undergone by proteins, such as fibrillogenesis. Fibrillation is common to many proteins, and in living organisms, it causes tissue-specific or systemic amyloid diseases. The nature of NPs and their surface chemistry is crucial in assessing their affinity for proteins and their effects on them. Here we present the first detailed structural characterization and molecular mechanics model of the interaction between a fibrillogenic protein, \u3b22-microglobulin, and a NP, 5 nm hydrophilic citrate-capped gold nanoparticles. NMR measurements and simulations at multiple levels (enhanced sampling molecular dynamics, Brownian dynamics, and Poisson-Boltzmann electrostatics) explain the origin of the observed protein perturbations mostly localized at the amino-terminal region. Experiments show that the protein-NP interaction is weak in the physiological-like, conditions and do not induce protein fibrillation. Simulations reproduce these findings and reveal instead the role of the citrate in destabilizing the lower pH protonated form of \u3b22-microglobulin. The results offer possible strategies for controlling the desired effect of NPs on the conformational changes of the proteins, which have significant roles in the fibrillation process

BLUU-Tramp data analysis: thermodynamic landscape of local unfolding processes

Isotopic exchange probed through NMR spectroscopy has been a precious source of information regarding local protein unfolding processes due to its ability to observe the exchange rate of single residues' amide sites. BLUU-Tramp extends this information allowing to follow, through two series of experiments, how the exchange constants vary over temperature. In this thesis a novel approach is introduced by means of an ad hoc calculation routine which is able to extract thermodynamic parameters from the decays of the amide signals, obtained through suitable treatment of the experimental data by another original routine (TinT) of the suite devised in our laboratory. By assuming that the isotopic exchange rates can be approximated with the EX2 limit, it is possible to estimate both the \u394G of local unfolding and other underlying thermodynamic parameters, in particular the \u394Cp. The method is used to obtain these parameters in two human proteins, \u3b22-microglobulin and lysozyme. Furthermore, a similar computational approach is presented which is able to extract thermodynamic parameters from classical isotopic exchange experiments carried out at various different temperatures. Comparisons among the results extracted from BLUU-Tramp series and classical exchange experiments are drawn and critical aspects in the latter kind of experiments are discusse