Biomedical subjects

Giovanni Settanni

Publications and source records attributed to Giovanni Settanni.

7 recordsLinked to original sources

PubMed2007

Estimation of folding probabilities and phi values from molecular dynamics simulations of reversible Peptide folding.

Molecular dynamics simulations with an implicit model of the solvent have allowed to investigate the reversible folding of structured peptides. For a 20-residue antiparallel beta-sheet peptide, the simulation results have revealed multiple folding pathways. Moreover, the conformational heterogeneity of the denatured state has been shown to originate from high enthalpy, high entropy basins with fluctuating non-native secondary structure, as well as low enthalpy, low entropy traps. An efficient and simple approach to estimate folding probabilities from molecular dynamics simulations has allowed to isolate conformations in the transition state ensemble and to evaluate Phi values, i.e., the effects of mutations on the folding kinetics and thermodynamic stability. These molecular dynamics studies have provided evidence that, if interpreted by neglecting the non-native interactions, Phi values overestimate the amount of native-like structure in the transition state.

Computer Simulation↗

PubMed2006

Unfolding transition state and intermediates of the tumor suppressor p16INK4a investigated by molecular dynamics simulations.

The ankyrin repeat is one of the most common protein motifs and is involved in protein-protein interactions. It consists of 33 residues that assume a beta-hairpin helix-loop-helix fold. Mutagenesis and kinetic experiments (Phi-value analysis of the folding transition state) have shown that the tumor suppressor p16(INK4a), a four-repeat protein, unfolds sequentially starting from the two N-terminal repeats. Here, the flexibility of p16(INK4a) at room temperature and its unfolding mechanism at high temperature have been investigated by multiple molecular dynamics runs in explicit water for a total simulation time of 0.65 micros. The transition state ensemble (TSE) of p16(INK4a) was identified by monitoring both the deviation from the experimental Phi values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid-body displacement of the first repeat involving both a rotation and translation is observed in all molecular dynamics simulations at high temperature. The Trp(15), Pro(75), and Ala(76) side-chains are more buried in the TSE than the native state. The sequential unfolding starting at the second repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding trajectories reveal the presence of two on-pathway intermediates with partial alpha-helical structure. Finally, on the basis of the available experimental and simulation results we suggest that in modular proteins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect.

Binding Sites↗

PubMed2005

Estimation of protein folding probability from equilibrium simulations.

The assumption that similar structures have similar folding probabilities (p(fold)) leads naturally to a procedure to evaluate p(fold) for every snapshot saved along an equilibrium folding-unfolding trajectory of a structured peptide or protein. The procedure utilizes a structurally homogeneous clustering and does not require any additional simulation. It can be used to detect multiple folding pathways as shown for a three-stranded antiparallel beta-sheet peptide investigated by implicit solvent molecular dynamics simulations.

Biophysics↗

PubMed2005

Phi-value analysis by molecular dynamics simulations of reversible folding.

In Phi-value analysis, the effects of mutations on the folding kinetics are compared with the corresponding effects on thermodynamic stability to investigate the structure of the protein-folding transition state (TS). Here, molecular dynamics (MD) simulations (totaling 0.65 ms) have been performed for a large set of single-point mutants of a 20-residue three-stranded antiparallel beta-sheet peptide. Between 57 and 120 folding events were sampled at near equilibrium for each mutant, allowing for accurate estimates of folding/unfolding rates and stability changes. The Phi values calculated from folding and unfolding rates extracted from the MD trajectories are reliable if the stability loss upon mutation is larger than approximately 0.6 kcal/mol, which is observed for 8 of the 32 single-point mutants. The same heterogeneity of the TS of the wild type was found in the mutated peptides, showing two possible pathways for folding. Single-point mutations can induce significant TS shifts not always detected by Phi-value analysis. Specific nonnative interactions at the TS were observed in most of the peptides studied here. The interpretation of Phi values based on the ratio of atomic contacts at the TS over the native state, which has been used in the past in MD and Monte Carlo simulations, is in agreement with the TS structures of wild-type peptide. However, Phi values tend to overestimate the nativeness of the TS ensemble, when interpreted neglecting the nonnative interactions.

Computer Simulation↗

PubMed2003

Molecular dynamics simulations of the NGF-TrkA domain 5 complex and comparison with biological data.

The nerve growth factor (NGF) is an important pharmacological target for Alzheimer's and other neurodegenerative diseases. Its action derives partly from its binding to the tyrosine kinase A receptor (TrkA). Here we study energetics and dynamics of the NGF-TrkA complex by carrying out multinanosecond molecular dynamics simulations, accompanied by electrostatic calculations based on the Poisson-Boltzmann equation. Our calculations, which are based on the x-ray structure of the complex, suggest that some of the mutations affecting dramatically the affinity of the complex involve residues that form highly favorable, direct or water-mediated hydrogen bond interactions at the ligand-receptor interface and, in some cases, that also critically participate to the large-scale motions of the complex. Furthermore, our calculations offer a rationale for the small effect on binding affinity observed upon specific mutations involving large changes in electrostatics (i.e., the charged-to-neutral mutations). Finally, these calculations, used along with the mutagenesis data, provide a basis for designing new peptides that mimic NGF in TrkA binding function.

Binding Sites↗

PubMed2002

The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies.

We describe the application of an intracellular antibody capture technology (IACT) as a generic in vivo selection procedure for isolating intracellular antibodies or ICAbs. IACT was applied to the de novo selection of functional ICAbs against the microtubule-associated protein TAU, found in neurofibrillary lesions of Alzheimer's disease brains. A panel of 17 different ICAbs was created which bind TAU inside cells and the epitopes recognized by the selected ICAbs have been determined by an in vivo epitope mapping procedure. Finally, sequence analysis showed that the IACT-derived ICAbs are characterized by a common signature of conserved amino acid residues, suggesting that the IACT naturally selects a sort of "captured consensus sequence" for intracellular antibodies. The development of IACT, together with the possibility of scaling up in a high throughput and automated format, makes IACT a new enabling tool for target validation in functional genomics and global proteomics.

Alzheimer Disease↗

PubMed2002

Folding pathways of prion and doppel.

The relevance of various residue positions for the stability and the folding characteristics of the prion protein in its normal cellular form are investigated by using molecular dynamics simulations of models exploiting the topology of the native state. These models allow for reproducing the experimentally validated two-state behavior of the normal prion isoform. Highly significant correlations are found between the most topologically relevant sites in our analysis and the single point mutations known to be associated with the arousal of the genetic forms of prion disease. Insight into the conformational change is provided by comparing the folding process of cellular prion and doppel that share a similar native state topology: the folding pathways of the former can be grouped in two main classes according to which tertiary structure contacts are formed first enroute to the native state. For the latter a single class of pathways leads to the native state again through a two-state process. Our results are consistent and supportive of the recent experimental findings that doppel lacks the scrapie isoform and that such remarkably different behavior involves residues in the region containing the two beta-strands and the intervening helix.

Amino Acids↗