The human immunodeficiency virus (HIV-1) matrix protein p17 (p17) is released from infected cells as a protein capable of deregulating the biological activity of different cells. P17 variants (vp17s), more frequently detected in the plasma of HIV-1+ patients with rather than without lymphoma and characterized by amino acids insertions in their C-terminal region, were found to trigger B cell growth and clonogenicity. Vp17s endowed with B-cell-growth-promoting activity are drastically destabilized, whereas, in a properly folded state, reference p17 (refp17) does not exert any biological activity on B cell growth and clonogenicity. However, misfolding of refp17 is necessary to expose a masked functional epitope, interacting with the protease-activated receptor 1 (PAR-1), endowed with B cell clonogenicity. Indeed, it is worth noting that changes in the secondary structure can strongly impact the function of a protein. Here, we performed computational studies to show that the gain of function of vp17s is linked to dramatic conformational changes due to structural modification in the secondary-structure elements and in the rearrangement of the hydrogen bond (H-bond) network. In particular, all clonogenic vp17s showed the disengagement of two critical residues, namely Trp16 and Tyr29, from their hydrophobic core. Biological data showed that the mutation of Trp16 and Tyr29 to Ala in the refp17 backbone, alone or in combination, resulted in a protein endowed with B cell clonogenic activity. These data show the pivotal role of the hydrophobic component in maintaining refp17 stability and identify a novel potential therapeutic target to counteract vp17-driven lymphomagenesis in HIV-1+ patients.

The p53 protein is a transcriptional regulatory factor and many of its functions require that it forms a tetrameric structure. Although the tetramerization domain of mammalian p53 proteins (p53TD) share significant sequence similarities, it was recently shown that the tree shrew p53TD is considerably more thermostable than the human p53TD. To determine whether other mammalian species display differences in this domain, we used biophysical, functional, and structural studies to compare the properties of the p53TDs from six mammalian model organisms (human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum). The results indicate that the p53TD from the opossum and tree shrew are significantly more stable than the human p53TD, and there is a correlation between the thermostability of the p53TDs and their ability to activate transcription. Structural analysis of the tree shrew and opossum p53TDs indicated that amino acid substitutions within two distinct regions of their p53TDs can dramatically alter hydrophobic packing of the tetramer, and in particular substitutions at positions corresponding to F341 and Q354 of the human p53TD. Together, the results suggest that subtle changes in the sequence of the p53TD can dramatically alter the stability, and potentially lead to important changes in the functional activity, of the p53 protein.

The structural transformation producing amyloids is a phenomenon that sheds new light on the protein folding problem. The analysis of the polymorphic structures of the α-synuclein amyloid available in the PDB database allows analysis of the amyloid-oriented structural transformation itself, but also the protein folding process as such. The polymorphic amyloid structures of α-synuclein analyzed employing the hydrophobicity distribution (fuzzy oil drop model) reveal a differentiation with a dominant distribution consistent with the micelle-like system (hydrophobic core with polar shell). This type of ordering of the hydrophobicity distribution covers the entire spectrum from the example with all three structural units (single chain, proto-fibril, super-fibril) exhibiting micelle-like form, through gradually emerging examples of local disorder, to structures with an extremely different structuring pattern. The water environment directing protein structures towards the generation of ribbon micelle-like structures (concentration of hydrophobic residues in the center of the molecule forming a hydrophobic core with the exposure of polar residues on the surface) also plays a role in the amyloid forms of α-synuclein. The polymorphic forms of α-synuclein reveal local structural differentiation with a common tendency to accept the micelle-like structuralization in certain common fragments of the polypeptide chain of this protein.

Metal-free bromoperoxidase BPO-A1 from Streptomyces aureofacience was selected among several similar enzymes exhibiting brominating activity as the most stable haloperoxidase against 70%(v/v) methanol. A comparison of the BPO-A1 and octahistidine-tagged BPO-A1 at the C-terminus (BPO-A1-His8) revealed that the His-tag enhanced the organic solvent-stability of BPO-A1 with pH- and heat-stabilities. Additionally, the contribution of the hydrophilicity at the C-terminal of BPO-A1 to the organic solvent-stability was confirmed employing several mutants bearing hydrophilic oligopeptides. Fortunately, two excellent mutants, BPO-A1-Lys8 and BPO-A1-Arg8, with high stabilities against various water-miscible organic solvents were obtained. In conclusion, the enhancing effect of the hydrophilic oligopeptides on the organic solvent-stability was associated with a decrease in the hydrophobic surface area near the C-terminus.

Numerous studies have investigated the differences and similarities between protein structures determined by solution NMR spectroscopy and those determined by X-ray crystallography. A fundamental question is whether any observed differences are due to differing methodologies or to differences in the behavior of proteins in solution versus in the crystalline state. Here, we compare the properties of the hydrophobic cores of high-resolution protein crystal structures and those in NMR structures, determined using increasing numbers and types of restraints. Prior studies have reported that many NMR structures have denser cores compared with those of high-resolution X-ray crystal structures. Our current work investigates this result in more detail and finds that these NMR structures tend to violate basic features of protein stereochemistry, such as small non-bonded atomic overlaps and few Ramachandran and sidechain dihedral angle outliers. We find that NMR structures solved with more restraints, and which do not significantly violate stereochemistry, have hydrophobic cores that have a similar size and packing fraction as their counterparts determined by X-ray crystallography at high resolution. These results lead us to conclude that, at least regarding the core packing properties, high-quality structures determined by NMR and X-ray crystallography are the same, and the differences reported earlier are most likely a consequence of methodology, rather than fundamental differences between the protein in the two different environments.

Cohesin holds sister chromatids together and is cleaved by separase/Cut1 to release DNA during the transition from mitotic metaphase to anaphase. The cohesin complex consists of heterodimeric structural maintenance of chromosomes (SMC) subunits (Psm1 and Psm3), which possess a head and a hinge, separated by long coiled coils. Non-SMC subunits (Rad21, Psc3 and Mis4) bind to the SMC heads. Kleisin/Rad21\'s N-terminal domain (Rad21-NTD) interacts with Psm3\'s head-coiled coil junction (Psm3-HCJ). Spontaneous mutations that rescued the cleavage defects in temperature-sensitive (ts) separase mutants were identified in the interaction interface, but the underlying mechanism is yet to be understood. Here, we performed site-directed random mutagenesis to introduce single amino acid substitutions in Psm3-HCJ and Rad21-NTD, and then identified 300 mutations that rescued the cohesin-releasing defects in a separase ts mutant. Mutational analysis indicated that the amino acids involved in hydrophobic cores (which may be in close contact) in Psm3-HCJ and Rad21-NTD are hotspots, since 80 mutations (approx. 27%) were mapped in these locations. Properties of these substitutions indicate that they destabilize the interaction between the Psm3 head and Rad21-NTD. Thus, they may facilitate sister chromatid separation in a cleavage-independent way through cohesin structural re-arrangement.

During the protein folding process in computer simulations involving the use of a United RESidue (UNRES) force field, an additional module was introduced to represent directly the presence of a polar solvent in water form. This module implements the fuzzy oil drop model (FOD) where the 3D Gauss function expresses the presence of a polar environment which directs the polypeptide chain folding process towards the generation of a centric hydrophobic core. Sample test polypeptide chains of 8 proteins with chain lengths ranging from 37 to 75 aa were simulated in silico using the UNRES (U) package with an implicit solvent model and a built-in module expressing the FOD model (UNRES-FOD-UNRES (U + F) interleaved simulation). The protein structure obtained by both *** simulation schemes, i.e., accordingly***U and U + F, for all the analyzed protein models shows the presence of a hydrophobic core including where it is absent in the native structure. The proposed FOD-M model (M-modified) explaining the source of this phenomenon reveals the need to modify the external field expressing the role of a folding environment. The modification takes into account the influence of other than polar factors present in the folding environment.

According to the \"jigsaw puzzle\" model of protein folding, the isomorphism between sequence and structure is substantially determined by the specific geometry of side-chain interactions, within the protein interior. In this work, we have attempted to predict the hydrophobic core of cyclophilin (LdCyp) from Leishmania donovani, utilizing a surface complementarity function, which selects for high goodness of fit between hydrophobic side-chain surfaces, rather in the manner of assembling a three-dimensional jigsaw puzzle. The computational core prediction method implemented here has been tried on two distinct scenarios, on the LdCyp polypeptide chain with native non-core residues and all core residues initially set to alanine, on a poly-glycine polypeptide chain. Molecular dynamics simulations appeared to indicate partial destabilization of the two designed sequences. However, experimental characterization of the designed sequences by circular dichroism (CD) spectroscopy and denaturant (GdmCl) induced unfolding, demonstrated disordered proteins. Stepwise reconstruction of the designed cores by cumulative sequential mutations identified the specific mutation (M122L) as primarily responsible for fold collapse and all design objectives were achieved upon rectifying this mutation. In summary, the study demonstrates regions of the core to contain highly specific (jigsaw puzzle-like) interactions sensitive to any perturbations and a predictive algorithm to identify such regions. A mutation within the core has been identified which exercises an inordinate influence on the global fold, reminiscent of metamorphic proteins. In addition, the computational procedure could predict substantial regions of the core (given main-chain coordinates) without any reference to non-core residues.

The natural environment of proteins is the polar aquatic environment and the hydrophobic (amphipathic) environment of the membrane. The fuzzy oil drop model (FOD) used to characterize water-soluble proteins, as well as its modified version FOD-M, enables a mathematical description of the presence and influence of diverse environments on protein structure. The present work characterized the structures of membrane proteins, including those that act as channels, and a water-soluble protein for contrast. The purpose of the analysis was to verify the possibility that an external force field can be used in the simulation of the protein-folding process, taking into account the diverse nature of the environment that guarantees a structure showing biological activity.

The beta-trefoil protein architecture is characterized by three repeating \"trefoil\" motifs related by rotational symmetry and postulated to have evolved via gene duplication and fusion events. Despite this apparent structural symmetry, the primary and secondary structural elements typically exhibit pronounced asymmetric features. A survey of this family of proteins has revealed that among the most conserved symmetric structural elements is a ubiquitous buried solvent which participates in a bridging H-bond with three different beta-strands in each of the trefoil motifs. A computational analysis reported that these waters are likely associated with a substantial enthalpic contribution to overall stability. In this report, a Pro mutation is used to disrupt one of the water H-bond interactions to a main chain amide, and the effects upon stability and folding kinetics are determined. Combined with Ala mutations, the separate effects upon side chain truncation and H-bond deletion are analyzed in terms of stability and folding kinetics. The results show that these buried waters act to assemble a central folding nucleus, and are responsible for ~20% of the overall favorable enthalpy of folding.