Structure and Thermal Stability of wtRop and RM6 Proteins through All-Atom Molecular Dynamics Simulations and Experiments.

In the current work we study, via molecular simulations and experiments, the folding and stability of proteins from the tertiary motif of 4-α-helical bundles, a recurrent motif consisting of four amphipathic α-helices packed in a parallel or antiparallel fashion. The focus is on the role of the loop region in the structure and the properties of the wild-type Rop (wtRop) and RM6 proteins, exploring the key factors which can affect them, through all-atom molecular dynamics (MD) simulations and supporting by experimental findings. A detailed investigation of structural and conformational properties of wtRop and its RM6 loopless mutation is presented, which display different physical characteristics even in their native states. Then, the thermal stability of both proteins is explored showing RM6 as more thermostable than wtRop through all studied measures. Deviations from native structures are detected mostly in tails and loop regions and most flexible residues are indicated. Decrease of hydrogen bonds with the increase of temperature is observed, as well as reduction of hydrophobic contacts in both proteins. Experimental data from circular dichroism spectroscopy (CD), are also presented, highlighting the effect of temperature on the structural integrity of wtRop and RM6. The central goal of this study is to explore on the atomic level how a protein mutation can cause major changes in its physical properties, like its structural stability.

Probing Protein Folding with Sequence-Reversed α-Helical Bundles.

Recurrent protein folding motifs include various types of helical bundles formed by α-helices that supercoil around each other. While specific patterns of amino acid residues (heptad repeats) characterize the highly versatile folding motif of four-α-helical bundles, the significance of the polypeptide chain directionality is not sufficiently understood, although it determines sequence patterns, helical dipoles, and other parameters for the folding and oligomerization processes of bundles. To investigate directionality aspects in sequence-structure relationships, we reversed the amino acid sequences of two well-characterized, highly regular four-α-helical bundle proteins and studied the folding, oligomerization, and structural properties of the retro-proteins, using Circular Dichroism Spectroscopy (CD), Size Exclusion Chromatography combined with Multi-Angle Laser Light Scattering (SEC-MALS), and Small Angle X-ray Scattering (SAXS). The comparison of the parent proteins with their retro-counterparts reveals that while the α-helical character of the parents is affected to varying degrees by sequence reversal, the folding states, oligomerization propensities, structural stabilities, and shapes of the new molecules strongly depend on the characteristics of the heptad repeat patterns. The highest similarities between parent and retro-proteins are associated with the presence of uninterrupted heptad patterns in helical bundles sequences.

Plant and Animal Innate Immunity Complexes: Fighting Different Enemies with Similar Weapons.

Both animals and plants express intracellular innate immunity receptors known as NLR (NOD-like receptors or nucleotide-binding domain and leucine-rich repeat receptors, respectively). For various mammalian systems, the specific formation of macromolecular structures, such as inflammasomes by activated NLR receptors, has been extensively reported. However, for plant organisms, the formation of such structures was an open scientific question for many years. This year, the first plant 'resistosome' structure was reported, revealing significant structural similarities to mammalian apoptosome and inflammasome structures. In this review, we summarize the key components comprising the mammalian apoptosome/inflammasome structures and the newly discovered plant resistosome, highlighting their commonalities and differences.

Expression, purification and crystallization of a protein resulting from the inversion of the amino-acid sequence of a helical bundle.

Earlier studies have found that the occurrence of inverse sequence identity in proteins is not indicative of three-dimensional similarity, but rather leads to different folds or unfolded proteins. Short helices, however, frequently keep their conformations when their sequences are inverted. To explore the impact of sequence inversion on long helices, revRM6, with the inverse amino-acid sequence relative to RM6, a highly stable variant of the ColE1 Rop protein, was engineered. RM6 is a highly regular four-α-helical bundle that serves as a model system for protein-folding studies. Here, the crystallization and preliminary crystallographic characterization of revRM6 are reported. The protein was overexpressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belonged to space group P41212, with unit-cell parameters a = b = 44.98, c = 159.74 Å, and diffracted to a resolution of 3.45 Å.

Structural plasticity of 4-α-helical bundles exemplified by the puzzle-like molecular assembly of the Rop protein.

The dimeric Repressor of Primer (Rop) protein, a widely used model system for the study of coiled-coil 4-α-helical bundles, is characterized by a remarkable structural plasticity. Loop region mutations lead to a wide range of topologies, folding states, and altered physicochemical properties. A protein-folding study of Rop and several loop variants has identified specific residues and sequences that are linked to the observed structural plasticity. Apart from the native state, native-like and molten-globule states have been identified; these states are sensitive to reducing agents due to the formation of nonnative disulfide bridges. Pro residues in the loop are critical for the establishment of new topologies and molten globule states; their effects, however, can be in part compensated by Gly residues. The extreme plasticity in the assembly of 4-α-helical bundles reflects the capacity of the Rop sequence to combine a specific set of hydrophobic residues into strikingly different hydrophobic cores. These cores include highly hydrated ones that are consistent with the formation of interchain, nonnative disulfide bridges and the establishment of molten globules. Potential applications of this structural plasticity are among others in the engineering of bio-inspired materials.