Current knowledge of structural biology is based mostly on studies in vitro. However, proteins and nucleic acids operate inside cells, where the concentration of macromolecules can be > 300 mg/mL. Hence, structural predictions of well-defined elements of added complexity might enhance our knowledge of cell biology processes. Our lab develops and uses a coarse-grain approach, which might help the modeling of very large macromolecular aggregates.
The cytoplasm includes all the material except the membrane and, in eukaryotic cells, the nucleus. It has far larger heterogeneity than water solutions, with a massive presence of nonspecific attractive and repulsive intermolecular interactions, in addition to volume exclusion. The cytoplasm is a highly saline solution, and this poses challenges for the ion models used in molecular simulation.
Fluorescence (Giepmans et al. Science 2006, 312, 217-224) and NMR spectroscopy (Sakakibara et al., Nature 2009, 458, 102-105)) are among the very few techniques that allow the observation of a particular protein in the sea of macromolecules present in the cell. Whilst for NMR is starting to provide already high-resolution structural information, only qualitative or semi-quantitative structural determinants can be obtained by fluorescence spectroscopy. In fact, the lack of our understanding of the physical origin of spectral features greatly hampers interpretation. Molecular simulation-based predictions could be of great help in this respect.
Ion models in physiological solutions
Several popular used ion models for molecular simulations of membrane channel have been parametrized at zero ionic strength. However, concentrations of ions are much higher in cell, and in particular inside biological ion channel or near DNA and RNA.
We are testing the commonly used ion models in term of the non-ideal properties of single ions in electrolyte solutions by free energy calculations (collaboration with
Bob Eisenberg and
Simone Raugei).
We plan also to test the domain of applicability of alkali ions’ force fields in describing ion permeation along the NanC porin (collaboration with
Bob Eisenberg group).
Reaction coordinate of ion permeation in the NanC porin
Towards protein aggregates
We are developing methods for the simulation and characterization of large systems which may be used for comparison with in cell studies. In particular, we are attempting at bridging the gap between time scales of feasible simulations and those of biologically relevant motions by developing a hybrid approach (MM/CG), in which a small biologically relevant region of the protein is treated at the level of detail allowed by classical MD (MM region), while the rest of the protein is treated at the course grained (CG) level.
An interface region bridges the discontinuity between full-atom and CG descriptions. We tested our method on two proteins for which MD simulations had been performed (HIV-1 protease and human beta-secretase). The MM/CG model is able to reproduce both the mesoscopic and local features of the protein dynamics. Because of its cheap computational cost (two order of magnitude faster than standard all-atom MD), the methodology might be useful for the investigation of molecular recognition events. We are now applying this method to large macromolecular aggregates such as the
pseudopilus from
E. Coli.
Computational spectroscopy
Optical properties of chromophores embody a key facet of cell biology, allowing for a precise interrogation of a variety of biochemical events, including signaling, metabolism, and aberrant processes. These range from probing transient interactions between biomolecules (proteins and
nucleic acids), to protein dynamics and fibrillation and plaque formation in neurodegenerative diseases. Understanding how the environment tunes such optical properties is therefore is therefore crucial, yet this information is so far mostly lacking. A powerful tool to address this issue is given by the so-called quantum mechanics/molecular mechanics (QM/MM) methods.
In this approach, the chromophore may be treated at the quantum mechanical level, while the environment is described with an effective potential: the influence of the MM (presumably very complex and very large) environment is basically included as an external potential and, in case
the chromophore is covalently bound to MM region, by a mechanical coupling with the environment.
Most often the QM approach is solved within density functional theory (DFT) to study ground state properties, and time-dependent DFT (TDDFT) when excited states are involved, as in the case of the optical properties TDDFT is computationally very efficient, yet its predictive power depends dramatically on the system and on the functional used to reproduce the exchange and correlation
interactions.
Several approaches, including post-Hartree-Fock ones (configuration interaction and similar methods), have been already used to predict optical properties of biomolecules.
Many-body perturbation theory (MBPT is an attractive alternative, although of course it comes with higher computational cost than TDDFT. However, biophysical applications of one of the most widely used schemes of MBPT, the combination of the GW method with the Bethe-Salpeter equation (BSE) are, so far, lacking. The GW method is used for the evaluation of the single quasiparticle energies, and the BSE to introduce excitonic effects. We are collaborating with
O. Pulci (University Tor Vergata, Rome) and
A. Rubio to establish protocols to provide accurate and transferable calculations of optical properties.
We are currently calculating the optical properties of rhodamine-based probes attached to the phosphate binding protein, which are used as inorganic phosphate (Pi) sensors. Binding of Pi to the protein provokes structural rearrangements of the protein. This is associated with a drastic change in the absorption and emission spectra of the rhodamines, along with an enhancement in the emission intensity of about 18 folds. The structural facets on which this process is based are not clear. Our calculations will be validated against experiments performed by the experimental collaborators in the
Martin Webb's group at the MRC in London. This work will establish the molecular basis of fluorescence and absorption spectral shifts upon ligand-used conformational changes and may be used as a general protocol in a variety of biophysical applications. This project has also been funded by European
DECI initiative granting access to the
DEISA supercomputing infrastructure and benefiting of the resource allocation of 600.000 cpu-hours.
