Investigating protein structure and dynamics through wide-angle X-ray solution scattering

Wide-angle X-ray scattering (WAXS) is a powerful tool that can be used to gain information on the structure and dynamics of proteins and other biomolecules in solution. Improved methods for the calculation of WAXS patterns from available or putative protein models allow to better exploit the structural information contained in the experimental data. These methods, together with recent applications of static and time-resolved WAXS, are briefly reviewed

Anharmonic activations in proteins and peptide model systems and their connection with supercooled water thermodynamics

Proteins, the nano-machines of living systems, are highly dynamic molecules. The time-scale of functionally relevant motions spans over a very broad range, from femtoseconds to several seconds. In particular, the pico- to nanoseconds region is characterized by side-chain and backbone anharmonic fluctuations that are responsible for many biological tasks like ligand binding, substrate recognition and enzymatic activity. Neutron scattering on hydrated protein powders reveals two main activations of anharmonic dynamics, characterized by different onset temperature and amplitude. Here we review our work on synthetic polypeptides, native proteins, and single amino acids to identify the physical origin of the two onsets—one involving water-independent local dynamics of methyl groups and, to a minor extent, of aromatic side-chains, and the other one, known as “protein dynamical transition”, concerning large scale functional protein fluctuations, most likely induced by a crossover in the structure and dynamics of hydration water connected with the second critical point hypothesis

Dynamics of nanoparticles in a supercooled liquid

The dynamic properties of nanoparticles suspended in a supercooled glass forming liquid are studied by x-ray photon correlation spectroscopy. While at high temperatures the particles undergo Brownian motion the measurements closer to the glass transition indicate hyperdiffusive behavior. In this state the dynamics is independent of the local structural arrangement of nanoparticles, suggesting a cooperative behavior governed by the near-vitreous solvent

Dynamical properties of water in living cells

With the aim of studying the effect of water dynamics on the properties of biological systems, in this paper, we present a quasi-elastic neutron scattering study on three different types of living cells, differing both in their morphological and tumor properties. The measured scattering signal, which essentially originates from hydrogen atoms present in the investigated systems, has been analyzed using a global fitting strategy using an optimized theoretical model that considers various classes of hydrogen atoms and allows disentangling diffusive and rotational motions. The approach has been carefully validated by checking the reliability of the calculation of parameters and their 99% confidence intervals. We demonstrate that quasi-elastic neutron scattering is a suitable experimental technique to characterize the dynamics of intracellular water in the angstrom/picosecond space/time scale and to investigate the effect of water dynamics on cellular biodiversity

Immobilization of proteins in silica gel: Biochemical and biophysical properties

The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulation within the pores of a silica gel three-dimensional network, the mechanism of interaction between the protein and the gel matrix, and its effects on protein structure, function, stability and dynamics. The main applications in the field of biosensor design are described. Special emphasis is devoted to silica gel encapsulation as a tool to selectively stabilize subsets of protein conformations for biochemical and biophysical studies, an application where silica-based encapsulation demonstrated superior performance with respect to other immobilization techniques

Functional and dysfunctional conformers of human neuroserpin Characterized by optical spectroscopies and molecular dynamics

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds

Brain lateralization probed by water diffusion at atomic-to-micrometric scale

Combined neutron scattering and diffusion nuclear magnetic resonance experiments have been used to reveal significant interregional asymmetries (lateralization) in bovine brain hemispheres in terms of myelin arrangement and water dynamics at micron to atomic scales. Thicker myelin sheaths were found in the left hemisphere using neutron diffraction. 4.7\u2009T dMRI and quasi-elastic neutron experiments highlighted significant differences in the properties of water dynamics in the two hemispheres. The results were interpreted in terms of hemisphere-dependent cellular composition (number of neurons, cell distribution, etc.) as well as specificity of neurological functions (such as preferential networking)

Application of MOOSY32 eNose to assess the Effects of Some Post Harvest Treatments on the Quality of "Salustiana" Orange Juice

A new prototype of Electronic Nose instrument, Multisensory Odor Olfactory System MOOSY32, with a processing method based on a multivariate classification analysis was used to assess different postharvest and storage treatments effects to Salustiana oranges. The analysis method is based on the measurement of the volatile compounds produced under different environmental and operational conditions. The Electronic Nose system revealed that orange juice flavor changes even when juices are analyzed right after each treatment and fruits are stored under refrigerated conditions. The instrument was able to detect even small changes in the aromatic pattern of the juices, confirming that the packing line itself is able to cause perceptible changes in the flavor. This can be a new and important finding in the Salustiana orange treatment that can lead to a significant improvement of fruits quality on the markets.Cupane, M.; Pelegrí Sebastiá, J.; Climent, E.; Guarrasi, V.; Sogorb Devesa, TC.; Germana, MA. (2015). Application of MOOSY32 eNose to assess the Effects of Some Post Harvest Treatments on the Quality of "Salustiana" Orange Juice. Journal of Biosensors and Bioelectronics. 6(4). doi:10.4172/2155-6210.1000184S6

Cupricyclins, Novel Redox-Active Metallopeptides Based on Conotoxins Scaffold

Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu2+ ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins

The Tempered Polymerization of Human Neuroserpin

Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation