Quantitative Measurement of Intrinsic GTP Hydrolysis for Carcinogenic Glutamine 61 Mutants in H-Ras.

Mutations of human oncoprotein p21H-Ras (hereafter "Ras") at glutamine 61 are known to slow the rate of guanosine triphosphate (GTP) hydrolysis and transform healthy cells into malignant cells. It has been hypothesized that this glutamine plays a role in the intrinsic mechanism of GTP hydrolysis by interacting with an active site water molecule that stabilizes the formation of the charged transition state at the γ-phosphate during hydrolysis. However, there is no comprehensive data set of the effects of mutations to Q61 on the protein's intrinsic catalytic rate, structure, or interactions with water at the active site. Here, we present the first comprehensive and quantitative set of initial rates of intrinsic hydrolysis for all stable variants of RasQ61X. We further conducted enhanced molecular dynamics (MD) simulations of each construct to determine the solvent accessible surface area (SASA) of the side chain at position 61 and compared these results to previously measured changes in electric fields caused by RasQ61X mutations. For polar and negatively charged residues, we found that the rates are normally distributed about an optimal electrostatic contribution, close to that of the native Q61 residue, and the rates are strongly correlated to the number of waters in the active site. Together, these results support a mechanism of GTP hydrolysis in which Q61 stabilizes a transient hydronium ion, which then stabilizes the transition state while the γ-phosphate is undergoing nucleophilic attack by a second, catalytically active water molecule. We discuss the implications of such a mechanism on future strategies for combating Ras-based cancers.

Towards mapping electrostatic interactions between Kdo2-lipid A and cationic antimicrobial peptides via ultraviolet photodissociation mass spectrometry.

Cationic antimicrobial peptides (CAMPs) have been known to act as multi-modal weapons against Gram-negative bacteria. As a new approach to investigate the nature of the interactions between CAMPs and the surfaces of bacteria, native mass spectrometry and two MS/MS strategies (ultraviolet photodissociation (UVPD) and higher energy collisional activation (HCD)) are used to examine formation and disassembly of saccharolipid·peptide complexes. Kdo2-lipid A (KLA) is used as a model saccharolipid to evaluate complexation with a series of cationic peptides (melittin and three analogs). Collisional activation of the KLA·peptide complexes results in the disruption of electrostatic interactions, resulting in apo-sequence ions with shifts in the distribution of ions compared to the fragmentation patterns of the apo-peptides. UVPD of the KLA·peptide complexes results in both apo- and holo-sequence ions of the peptides, the latter in which the KLA remains bound to the truncated peptide fragment despite cleavage of a covalent bond of the peptide backbone. Mapping both the N- and C-terminal holo-product ions gives insight into the peptide motifs (specifically an electropositive KRKR segment and a proline residue) that are responsible for mediating the electrostatic interactions between the cationic peptides and saccharolipid.

Development of a sustainability technical guide for the Italian olive oil supply chain.

Italian olive growing must aim at the transition to economically and environmentally sustainable management systems, linked to premium quality production and to a recognized and remunerated context of biodiversity conservation in compliance with the provisions of the European Union New Green Deal and United Nations Agenda 2030. To assist and facilitate companies in this step, a sustainability technical guide for the Italian olive oil supply chain has been developed, with reference to the four pillars of sustainability. The guide, consisting of 42 requirements, was submitted to 18 olive farms from 8 different Italian regions participating in this pilot study, to assess their level of total sustainability and to receive feedbacks throughout the drafting process. Taken as a whole, the companies have proved to be virtuous in meeting the requirements provided, with percentages of compliance ranging from 86 to 96% according to pillar and from 70 to 100% according to company and showed a remarkable spirit of collaboration and involvement in the construction of the guide. In this regard, the text is aimed to represent a participatory standard for Italian institutions and for other olive countries.

Beyond pKa: Experiments and Simulations of Nitrile Vibrational Probes in Staphylococcal Nuclease Show the Importance of Local Interactions.

Electric fields are fundamentally important to biological phenomena, but are difficult to measure experimentally or predict computationally. Changes in pKa of titratable residues have long been used to report on local electrostatic fields in proteins. Alternatively, nitrile vibrational probes are potentially less disruptive and more direct reporters of local electrostatic field, but quantitative interpretation is clouded by the ability of the nitrile to accept a hydrogen bond. To this end, we incorporated nitrile probes into 10 locations of staphylococcal nuclease (SNase) where pKa shifts had already been determined. We characterized the local environment of each nitrile probe experimentally, through temperature-dependent spectroscopy, and computationally, through molecular dynamics simulations, and show that hydrogen bonding interactions dominate the spectral line shapes. We demonstrate that the information provided by the line shape of the nitrile spectra, compared to scalar values of pKa shift or nitrile frequency shift, better describes local environments in proteins in a manner that will be useful for future computational efforts to predict electrostatics in complex biological systems.

Structural Evaluation of Protein/Metal Complexes via Native Electrospray Ultraviolet Photodissociation Mass Spectrometry.

Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational modifications, but also their tertiary structures. In this study, three metal-binding proteins, Staphylococcal nuclease, azurin, and calmodulin, are used to demonstrate the use of UVPD to elucidate metal-binding regions via comparisons between the fragmentation patterns of apo (metal-free) and holo (metal-bound) proteins. The binding of staphylococcal nuclease to calcium was evaluated, in addition to a series of lanthanide(III) ions which are expected to bind in a similar manner as calcium. On the basis of comparative analysis of the UVPD spectra, the binding region for calcium and the lanthanide ions was determined to extend from residues 40-50, aligning with the known crystal structure. Similar analysis was performed for both azurin (interrogating copper and silver binding) and calmodulin (four calcium binding sites). This work demonstrates the utility of UVPD methods for determining and analyzing the metal binding sites of a variety of classes of proteins.

Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry.

Mutations in the GTPase enzyme K-Ras, specifically at codon G12, remain the most common genetic alterations in human cancers. The mechanisms governing activation of downstream signaling pathways and how they relate back to the identity of the mutation have yet to be completely defined. Here we use native mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) to investigate the impact of three G12X mutations (G12C, G12V, G12S) on the homodimerization of K-Ras as well as heterodimerization with a downstream effector protein, Raf. Electrospray ionization (ESI) was used to transfer complexes of WT or G12X K-Ras bound to guanosine 5'-diphosphate (GDP) or GppNHp (non-hydrolyzable analogue of GTP) into the gas phase. Relative abundances of homo- or hetero-dimer complexes were estimated from ESI-MS spectra. K-Ras + Raf heterocomplexes were activated with UVPD to probe structural changes responsible for observed differences in the amount of heterocomplex formed for each variant. Holo (ligand-bound) fragment ions resulting from photodissociation suggest the G12X mutants bind Raf along the expected effector binding region (ß-interface) but may interact with Raf via an alternative α-interface as well. Variations in backbone cleavage efficiencies during UV photoactivation of each variant were used to relate mutation identity to structural changes that might impact downstream signaling. Specifically, oncogenic upregulation for hydrogen-bonding amino acid substitutions (G12C, G12S) is achieved by stabilizing ß-interface interactions with Raf, while a bulkier, hydrophobic G12V substitution leads to destabilization of this interface and instead increases the proximity of residues along the α-helical bundles. This study deciphers new pieces of the complex puzzle of how different K-Ras mutations exert influence in downstream signaling.

A new approach to assessing the water footprint of wine: an Italian case study.

Agriculture is the largest freshwater consumer, accounting for 70% of the world's water withdrawal. Water footprints (WFs) are being increasingly used to indicate the impacts of water use by production systems. A new methodology to assess WF of wine was developed in the framework of the V.I.V.A. project (Valutazione Impatto Viticoltura sull'Ambiente), launched by the Italian Ministry for the Environment in 2011 to improve the Italian wine sector's sustainability. The new methodology has been developed that enables different vines from the same winery to be compared. This was achieved by calculating the gray water footprint, following Tier III approach proposed by Hoekstra et al. (2011). The impact of water use during the life cycle of grape-wine production was assessed for six different wines from the same winery in Sicily, Italy using both the newly developed methodology (V.I.V.A.) and the classical methodology proposed by the Water Footprint Network (WFN). In all cases green water was the largest contributor to WF, but the new methodology also detected differences between vines of the same winery. Furthermore, V.I.V.A. methodology assesses water body contamination by pesticides application whereas the WFN methodology considers just fertilization. This fact ended highlights the highest WF of vineyard 4 calculated by V.I.V.A. if compared with the WF calculated with WFN methodology. Comparing the WF of wine produced with grapes from the six different wines, the factors most greatly influencing the results obtained in this study were: distance from the water body, fertilization rate, amount and eco-toxicological behavior of the active ingredients used.