Atomic structural details of a protein grafted onto gold nanoparticles

Abstract The development of a methodology for the structural characterization at atomic detail of proteins conjugated to nanoparticles would be a breakthrough in nanotechnology. Solution and solid-state NMR spectroscopies are currently used to investigate molecules and peptides grafted onto nanoparticles, but the strategies used so far fall short in the application to proteins, which represent a thrilling development in theranostics. We here demonstrate the feasibility of highly-resolved multidimensional heteronuclear spectra of a large protein assembly conjugated to PEGylated gold nanoparticles. The spectra have been obtained by direct proton detection under fast MAS and allow for both a fast fingerprinting for the assessment of the preservation of the native fold and for resonance assignment. We thus demonstrate that the structural characterization and the application of the structure-based methodologies to proteins bound to gold nanoparticles is feasible and potentially extensible to other hybrid protein-nanomaterials

1 H-detected solid-state NMR of proteins entrapped in bioinspired silica: A new tool for biomaterials characterization

Proton-detection in solid-state NMR, enabled by high magnetic fields (>18 T) and fast magic angle spinning (>50 kHz), allows for the acquisition of traditional (1)H-(15)N experiments on systems that are too big to be observed in solution. Among those, proteins entrapped in a bioinspired silica matrix are an attractive target that is receiving a large share of attention. We demonstrate that (1)H-detected SSNMR provides a novel approach to the rapid assessment of structural integrity in proteins entrapped in bioinspired silica

Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques

With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.Microbial Biotechnolog

Automated Determination of Nuclear Magnetic Resonance Chemical Shift Perturbations in Ligand Screening Experiments:The PICASSO Web Server

[Image: see text] Nuclear magnetic resonance (NMR) is an effective, commonly used experimental approach to screen small organic molecules against a protein target. A very popular method consists of monitoring the changes of the NMR chemical shifts of the protein nuclei upon addition of the small molecule to the free protein. Multidimensional NMR experiments allow the interacting residues to be mapped along the protein sequence. A significant amount of human effort goes into manually tracking the chemical shift variations, especially when many signals exhibit chemical shift changes and when many ligands are tested. Some computational approaches to automate the procedure are available, but none of them as a web server. Furthermore, some methods require the adoption of a fairly specific experimental setup, such as recording a series of spectra at increasing small molecule:protein ratios. In this work, we developed a tool requesting a minimal amount of experimental data from the user, implemented it as an open-source program, and made it available as a web application. Our tool compares two spectra, one of the free protein and one of the small molecule:protein mixture, based on the corresponding peak lists. The performance of the tool in terms of correct identification of the protein-binding regions has been evaluated on different protein targets, using experimental data from interaction studies already available in the literature. For a total of 16 systems, our tool achieved between 79% and 100% correct assignments, properly identifying the protein regions involved in the interaction

G-triplex structure and formation propensity

The occurrence of a G-triplex folding intermediate of thrombin binding aptamer (TBA) has been recently predicted by metadynamics calculations, and experimentally supported by Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) data collected on a 3′ end TBA-truncated 11-mer oligonucleotide (11-mer-3′-t-TBA). Here we present the solution structure of 11-mer-3′-t-TBA in the presence of potassium ions. This structure is the first experimental example of a G-triplex folding, where a network of Hoogsteen-like hydrogen bonds stabilizes six guanines to form two G:G:G triad planes. The G-triplex folding of 11-mer-3′-t-TBA is stabilized by the potassium ion and destabilized by increasing the temperature. The superimposition of the experimental structure with that predicted by metadynamics shows a great similarity, with only significant differences involving two loops. These new structural data show that 11-mer-3′-t-TBA assumes a G-triplex DNA conformation as its stable form, reinforcing the idea that G-triplex folding intermediates may occur in vivo in human guanine-rich sequences. NMR and CD screening of eight different constructs obtained by removing from one to four bases at either the 3′ and the 5′ ends show that only the 11-mer-3′-t-TBA yields a relatively stable G-triple

Real-Time Insights into Biological Events: In-Cell Processes and Protein-Ligand Interactions

FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies