α-Hydrazino Acid Insertion Governs Peptide Organization in Solution by Local Structure Ordering.

In this work, we have applied the concept of α-hydrazino acid insertion in a peptide sequence as a means of structurally organizing a potential protein-protein interactions (PPI) inhibitor. Hydrazino peptides characterized by the incorporation of an α-hydrazino acid at specific positions introduce an additional nitrogen atom into their backbone. This modification leads to a change in the electrostatic properties of the peptide and induces the restructuring of its hydrogen bonding network, resulting in conformational changes toward more stable structural motifs. Despite the successful use of synthetic hydrazino oligomers in binding to nucleic acids, the structural changes due to the incorporation of α-hydrazino acid into short natural peptides in solution are still poorly understood. Based on NMR data, we report structural models of p53-derived hydrazino peptides with elements of localized peptide structuring in the form of an α-, ß-, or γ-turn as a result of hydrazino modification in the peptide backbone. The modifications could potentially lead to the preorganization of a helical secondary peptide structure in a solution that is favorable for binding to a biological receptor. Spectroscopically, we observed that the ensemble averaged rapidly interconverting conformations, including isomerization of the E-Z hydrazide bond. This further increases the adaptability by expanding the conformational space of hydrazine peptides as potential protein-protein interaction antagonists.

From structural polymorphism to structural metamorphosis of the coat protein of flexuous filamentous potato virus Y.

The structural diversity and tunability of the capsid proteins (CPs) of various icosahedral and rod-shaped viruses have been well studied and exploited in the development of smart hybrid nanoparticles. However, the potential of CPs of the wide-spread flexuous filamentous plant viruses remains to be explored. Here, we show that we can control the shape, size, RNA encapsidation ability, symmetry, stability and surface functionalization of nanoparticles through structure-based design of CP from potato virus Y (PVY). We provide high-resolution insight into CP-based self-assemblies, ranging from large polymorphic or monomorphic filaments to smaller annular, cubic or spherical particles. Furthermore, we show that we can prevent CP self-assembly in bacteria by fusion with a cleavable protein, enabling controlled nanoparticle formation in vitro. Understanding the remarkable structural diversity of PVY CP not only provides possibilities for the production of biodegradable nanoparticles, but may also advance future studies of CP's polymorphism in a biological context.

Generation and in Planta Functional Analysis of Potato Virus Y mutants.

Potato virus Y (PVY), the type member of the genus Potyvirus (family Potyviridae), is the most widespread virus affecting potato and is included in the top five most economically detrimental plant viruses. Recently, the structure of the PVY virion has been determined by cryo-electron microscopy, which has opened the doors to functional studies that explore the involvement of selected amino acids in different stages of the viral cycle. The only way to functionally challenge in planta the role of particular amino acids in the coat protein of PVY, or in other viral proteins, is by using cDNA clones. The use and manipulation of PVY cDNA clones, unlike those of other potyviruses, has been traditionally impaired by the toxicity that certain sequences within the PVY genome pose to Escherichia coli. Here, we describe the use of a published PVY cDNA clone, which harbours introns that overcome the aforementioned toxicity, to explore the effects of different coat protein modifications on viral infection. The protocol includes manipulation of the cDNA clone in E. coli, biolistic inoculation of plants with the constructed clones, observation of the biological effects on plants, quantification of cDNA clones by reverse transcription quantitative PCR, and confirmation of virion formation by transmission electron microscopy. Future possibilities involve the use of PVY cDNA clones tagged with fluorescent protein reporters to allow further insights into the effects of coat protein mutations on the cell-to-cell movement of PVY virions.

Structural basis for the multitasking nature of the potato virus Y coat protein.

Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY's flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA-coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein's amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.