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The 20th IUPAB Congress took place online, together with the annual meetings of the Brazilian Biophysical Society and the Brazilian Society for Biochemistry and Molecular Biology, from the 4th to the 8th of October, 2021. The ten keynote lectures, 24 symposia, two poster sessions, and a series of technical seminars covered the full diversity of current biophysical research and its interfaces with other fields. The event had over 1000 attendees, with an excellent gender balance. Although the Americas dominated, there were also significant numbers of participants from Europe, Asia, and Africa.

The relationship between GTP binding and hydrolysis on the one hand and septin filament assembly and function on the other has yet to be fully elucidated. The crystal structures of SEPT3 subgroup members bound to either GDP or to a GTP analogue shed light on this question by indicating how the squeezing of one of the inter-subunit interfaces may lead to the exposure of a polybasic region important for membrane association., Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.

Multi-drug resistance (MDR) is a serious threat to global public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in depth biochemical, microbicidal and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (Im) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. Probably the integrity of the H-N-H motif is essential in the killing activity of S8, as Glu100 is a highly conserved component of this structure. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala pyocin-S8DNase-Im complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possible use of S8 as an alternative antibiotic for MDR Pseudomonas aeruginosa strains, while leaving commensal human microbiota intact.

Septins are GTP binding proteins considered to be a novel component of the cytoskeleton. They polymerize into filaments based on hetero-oligomeric core particles which, in humans, are either hexamers or octamers composed of two copies each of either three or four different septins from the 13 available. Not all combinations are possible as it is believed that these must obey substitution rules which determine that different septins must be derived from four distinct and well-established sub-groups. Here, we have purified and characterized one such combinations, SEPT5-SEPT6-SEPT7, and used TEM to derive the first structural information concerning its assembly. The full complex was purified using an affinity tag attached to only one of its components (SEPT7) and was able to bind to and perturb lipid bilayers. Although the complex assembled into elongated hexameric particles, the position of SEPT5 was incompatible with that predicted by the reported structure of SEPT2-SEPT6-SEPT7 based on the substitution rules. MBP-fusion constructs for SEPT5 and SEPT2 and immuno-staining clearly show that these septins occupy the terminal positions of the SEPT5-SEPT6-SEPT7 and SEPT2-SEPT6-SEPT7 hexamers, respectively. In so doing they expose a so-called NC interface which we show to be more susceptible to perturbation at high salt concentrations. Our results show that the true structure of the hexamer is inverted with respect to that described previously and, as such, is more compatible with that reported for yeast. Taken together, our results suggest that the mechanisms involved in spontaneous self-assembly of septin core particles and their filaments deserve further reflection.

Superoxide dismutase-1 (SOD1) maturation comprises a string of posttranslational modifications which transform the nascent peptide into a stable and active enzyme. The successive folding, metal ion binding, and disulphide acquisition steps in this pathway can be catalysed through a direct interaction with the copper chaperone for SOD1 (CCS). This process confers enzymatic activity and reduces access to noncanonical, aggregation-prone states. Here, we present the functional mechanisms of human copper chaperone for SOD1 (hCCS)–catalysed SOD1 activation based on crystal structures of reaction precursors, intermediates, and products. Molecular recognition of immature SOD1 by hCCS is driven by several interface interactions, which provide an extended surface upon which SOD1 folds. Induced-fit complexation is reliant on the structural plasticity of the immature SOD1 disulphide sub-loop, a characteristic which contributes to misfolding and aggregation in neurodegenerative disease. Complexation specifically stabilises the SOD1 disulphide sub-loop, priming it and the active site for copper transfer, while delaying disulphide formation and complex dissociation. Critically, a single destabilising amino acid substitution within the hCCS interface reduces hCCS homodimer affinity, creating a pool of hCCS available to interact with immature SOD1. hCCS substrate specificity, segregation between solvent and biological membranes, and interaction transience are direct results of this substitution. In this way, hCCS-catalysed SOD1 maturation is finessed to minimise copper wastage and reduce production of potentially toxic SOD1 species., Author summary Cellular complexity necessitates an equally complex network of courier proteins to internalise, sort, and deliver biologically useful metals like copper. These relay systems negotiate a landscape of metal-binding sites through handshake–handoff interactions, but the mechanisms that impart a necessary transience are often not clear. Superoxide dismutase-1 (SOD1) is one of the most abundant human proteins and is an important part of our antioxidant, redox signalling and respiratory control mechanisms. If newly synthesised SOD1 is not correctly processed by the addition of copper, zinc, and an unusual disulphide bond, it will remain inactive or can misfold, as is the case in some neurodegenerative diseases. Here, we discover the mechanisms that govern SOD1 maturation and stabilisation through interaction with the chaperone protein hCCS. Conservation of our proposed mechanism across eukaryotes indicates it developed very soon after the gene duplication event that separated SOD1 and CCS coding sequences. SOD1 stability appears to have been quickly traded at the expense of CCS following the dawn of eukaryotic life, in order to efficiently produce this important enzyme.

Halophilic organisms inhabit hypersaline environments where the extreme ionic conditions and osmotic pressure have driven the evolution of molecular adaptation mechanisms. Understanding such mechanisms is limited by the common difficulties encountered in cultivating such organisms. Within the Euryarchaeota, for example, only the Halobacteria and the order Methanosarcinales include readily cultivable halophilic species. Furthermore, only the former have been extensively studied in terms of their component proteins. Here, in order to redress this imbalance, we investigate the halophilic adaptation of glycolytic enzymes from the ADP-dependent phosphofructokinase/glucokinase family (ADP-PFK/GK) derived from organisms of the order Methanosarcinales. Structural analysis of proteins from non-halophilic and halophilic Methanosarcinales shows an almost identical composition and distribution of amino acids on both the surface and within the core. However, these differ from those observed in Halobacteria or Eukarya. Proteins from Methanosarcinales display a remarkable increase in surface lysine content and have no reduction to the hydrophobic core, contrary to the features ubiquitously observed in Halobacteria and which are thought to be the main features responsible for their halophilic properties. Biochemical characterization of recombinant ADP-PFK/GK from M. evestigatum (halophilic), and M. mazei (non-halophilic) shows the activity of both these extant enzymes to be only moderately inhibited by salt. Nonetheless, its activity over time is notoriously stabilized by salt. Furthermore, glycine betaine has a protective effect against KCl inhibition and enhances the thermal stability of both enzymes. The resurrection of the last common ancestor of ADP-PFK/GK from Methanosarcinales shows that the ancestral enzyme displays an extremely high salt tolerance and thermal stability. Structure determination of the ancestral protein reveals unique traits such as an increase in the Lys and Glu content at the protein surface and yet no reduction to the volume of the hydrophobic core. Our results suggest that the halophilic character is an ancient trait in the evolution of this protein family and that proteins from Methanosarcinales have adapted to highly saline environments by a non-canonical strategy, different from that currently proposed for Halobacteria. These results open up new avenues for the search and development of novel salt tolerant biocatalysts.

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.

Septins are able to polymerize into long apolar filaments and have long been considered to be a component of the cytoskeleton alongside intermediate filaments (which are also apolar in nature), microtubules and actin filaments (which are not). Their central guanosine triphosphate (GTP)-binding domain, which is essential for stabilizing the filament itself, is flanked by N- and C-terminal domains for which no direct structural information is yet available. In most cases, physiological filaments are built from a number of different septin monomers, and in the case of mammalian septins this is most commonly either three or four. Comprehending the structural basis for the spontaneous assembly of such filaments requires a deeper understanding of the interfaces between individual GTP-binding domains than is currently available. Nevertheless, in this review we will summarize the considerable progress which has been made over the course of the last 10 years. We will provide a brief description of each structure determined to date and comment on how it has added to the body of knowledge which is rapidly growing. Rather than simply repeat data which have already been described in the literature, as far as is possible we will try to take advantage of the full set of information now available (mostly derived from human septins) and draw the reader's attention to some of the details of the structures themselves and the filaments they form which have not be commented on previously. An additional aim is to clarify some misconceptions.

Cold shock proteins (Csp) constitute a family of ubiquitous small proteins that act as RNA-chaperones to avoid cold-induced termination of translation. All members contain two subdomains composed of 2 and 3 β-strands, respectively, which are connected by a hinge loop and fold into a β-barrel. Bacillus caldolyticus Csp (BcCsp) is one of the most studied members of the family in terms of its folding, function, and structure. This protein has been described as a monomer in solution, although a recent crystal structure showed dimerization via domain swapping (DS). In contrast, other cold shock proteins of the same fold are known to dimerize in a nonswapped arrangement. Hypothesizing that reducing the size of the hinge loop may promote swapping as in several other DS proteins with different folds we deleted two residues from these region (BcCsp∆36-37), leading to a protein in monomer–dimer equilibrium with similar folding stability to that of the wild-type. Strikingly, the crystal structure of BcCsp∆36-37 revealed a nonswapped dimer with its interface located at the nucleic acid-binding surface, showing that the deletion led to structural consequences far from the perturbation site. Concomitantly, circular dichroism experiments on BcCsp∆36-37 demonstrated that binding of the oligonucleotide hexathymidine disrupts the dimer. Additionally, HDXMS shows a protective effect on the protein structure upon dimerization, where the resulting interactions between ligand-binding surfaces in the dimer reduced the extent of exchange throughout the whole protein. Our work provides evidence of the complex interplay between conformational dynamics, deletions, and oligomerization within the Csp protein family. Databases Structural data are available in the Protein Data Bank under accession number 5JX4.

This paper explores the structural continuum in CATH and the extent to which superfamilies adopt distinct folds. Although most superfamilies are structurally conserved, in some of the most highly populated superfamilies (4% of all superfamilies) there is considerable structural divergence. While relatives share a similar fold in the evolutionary conserved core, diverse elaborations to this core can result in significant differences in the global structures. Applying similar protocols to examine the extent to which structural overlaps occur between different fold groups, it appears this effect is confined to just a few architectures and is largely due to small, recurring super-secondary motifs (e.g., alphabeta-motifs, alpha-hairpins). Although 24% of superfamilies overlap with superfamilies having different folds, only 14% of nonredundant structures in CATH are involved in overlaps. Nevertheless, the existence of these overlaps suggests that, in some regions of structure space, the fold universe should be seen as more continuous.

The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na(+). Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K(+) or Cs(+). Kinetic characterization of the enzyme shows that K(+) and Na(+) alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K(+) (but not Na(+)) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K(+) (but not Na(+)) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs(+) or K(+). These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.

Made available in DSpace on 2013-08-28T14:09:29Z (GMT). No. of bitstreams: 1 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) Made available in DSpace on 2013-09-30T18:37:20Z (GMT). No. of bitstreams: 2 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) WOS000298666200001.pdf.txt: 56655 bytes, checksum: 10d0e581654ecca834117e332f3f69ca (MD5) Previous issue date: 2011-12-20 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:49:40Z No. of bitstreams: 2 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) WOS000298666200001.pdf.txt: 56655 bytes, checksum: 10d0e581654ecca834117e332f3f69ca (MD5) Made available in DSpace on 2014-05-20T13:49:40Z (GMT). No. of bitstreams: 2 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) WOS000298666200001.pdf.txt: 56655 bytes, checksum: 10d0e581654ecca834117e332f3f69ca (MD5) Previous issue date: 2011-12-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Correlated mutation analysis has a long history of interesting applications, mostly in the detection of contact pairs in protein structures. Based on previous observations that, if properly assessed, amino acid correlation data can also provide insights about functional sub-classes in a protein family, we provide a complete framework devoted to this purpose. An amino acid specific correlation measure is proposed, which can be used to build networks summarizing all correlation and anti-correlation patterns in a protein family. These networks can be submitted to community structure detection algorithms, resulting in subsets of correlated amino acids which can be further assessed by specific parameters and procedures that provide insight into the relationship between different communities, the individual importance of community members and the adherence of a given amino acid sequence to a given community. By applying this framework to three protein families with contrasting characteristics (the Fe/Mn-superoxide dismutases, the peroxidase-catalase family and the C-type lysozyme/alpha-lactalbumin family), we show how our method and the proposed parameters and procedures are related to biological characteristics observed in these protein families, highlighting their potential use in protein characterization and gene annotation. Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil Univ São Paulo, Inst Fis Sao Carlos, Dept Fis & Informat, Sao Carlos, SP, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil FAPESP: 08/58734-1 FAPESP: 98/14138-2

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer–dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli.

Escherichia coli contains two phosphofructokinases, Pfk-1 and Pfk-2, which belong to unrelated protein families. In addition to catalytic function, the enzymes have converged in showing substrate inhibition by the nucleotide MgATP. However, although both Pfk-1 and Pfk-2 have been extensively characterized biochemically, only the structure of the former has been solved by X-ray diffraction. In order to fully understand how the same function has evolved on different structural folds, Pfk-2 has been crystallized by the hanging-drop vapour-diffusion method using PEG 6000 as precipitant. Single crystals were grown in the presence of MgATP and diffracted to 1.98 A. The crystals belong to the orthorhombic system, space group P222(1), with unit-cell parameters a = 42.8, b = 86.8, c = 171.3 A. The calculated Matthews coefficient of 2.45 A(3) Da(-1) indicates the presence of two monomers in the asymmetric unit, corresponding to a solvent content of 49%. Structure determination is ongoing.

The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.

Staphylococcal scalded-skin syndrome is usually diagnosed clinically by its characteristic exfoliating rash. Isolation of Staphylococcus aureus from the patient further supports the diagnosis. Several detection systems have been developed to determine whether the isolated strain produces exfoliative toxin, but none are routinely available in hospital laboratories. In a novel approach, we used computer models to predict the structure of the exfoliative toxins based on other serine proteases and to identify surface epitopes for the production of antibodies that specifically bound the exfoliative toxin A (ETA) serotype. Several rapid immunologically based diagnostic tests for ETA were developed with these antibodies and compared with existing systems. Our results showed that Western blot analysis using these antibodies was in complete correlation with PCR, which has been validated against the “gold standard” mouse model. On the other hand, the double-antibody enzyme-linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion assay gave unacceptably high false-positive results due to interference by staphylococcal protein A. This problem was successfully overcome by the development of a F(ab′) 2 fragment ELISA, which was rapid and reproducible and was as sensitive and specific as PCR and Western blot analysis. The F(ab′) 2 fragment ELISA is superior to existing diagnostic systems because it is quantitative, which may be related to the severity of the condition, and can detect amounts of exfoliative toxin in the picogram range directly from serum. This is the first detection system with the potential to confirm the diagnosis of staphylococcal scalded-skin syndrome from a routine blood test within 3 h of presentation.

Secondary structure prediction parameters and optimised decision constants for use with the method of Garnier et al. [(1978) J. Mol. Biol. 120, 97-120] have been derived for two new and distinct substates of beta-structure. These we term internal and external on the basis of their hydrogen bonding patterns. The profiles of the amino acids for several of the parameters are considerably different in the two substates. Predictions using the new parameters attempt to distinguish the strands at the core of the beta-sheet from those at its edges and so restrict the possible topologies in tertiary structure prediction. The potential application of these parameters is illustrated for the class of beta/alpha proteins.

Certain strains of Staphylococcus aureus usually belonging to phage group II produce epidermolytic toxins (ETA and ETB) which cause intraepidermal splitting in mice, neonates and occasionally adults. Amino acid sequences of ETA and ETB have been reported but the mechanism of epidermolysis remains unknown. A search of the NBRF-PIR computer database showed the toxins to have significant sequence similarity with staphylococcal V8 protease and that the catalytic triad of V8 protease is present in ETA and ETB. Comparison of ETA, ETB and V8 protease with other members of the trypsin-like serine protease family revealed little homology save for the immediate vicinity of the residues constituting the catalytic triad. The toxins, therefore, exhibit a distant relationship to mammalian serine proteases. A potential Ca2+-binding loop was identified in ETA (but not ETB) on the basis of sequence similarity with the second calcium-binding loop of rat intestinal calcium-binding protein. Epidermolysis produced by ETA in the mouse bioassay was shown to be inhibited by the presence of EDTA consistent with a Ca2+-dependent mechanism.

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.

GRASPs (from Golgi ReAssembly and Stacking Proteins) are proteins involved in the organization and maintenance of the Golgi complex. While the extension of this role has been questioned in the past few years, many other functions have been assigned to them. In particular, we highlight the participation in tethering vesicles that need to move along the Golgi and processes of unconventional protein secretion. Structurally, GRASPs can be divided in an N-terminal domain called GRASP and a non-conserved, highly disordered C-terminal domain, termed SPR. Structural information, hitherto, has been scarce, which motivated the beginning of this project. To study GRASPs, we used the only GRASP (called Grh1) of a model organism: the yeast Saccharomyces cereviseae. We showed that Grh1 contains regions of intrinsic disorder also in its GRASP domain, being classified as a molten globule. Besides, Grh1 is capable of forming amyloid-like fibrils when in specific conditions in vitro, such as low pH and moderately elevated temperature. With the aim of investigating a possible relationship between fibril formation and the role played by Grh1 in unconventional protein secretion, part of the present thesis was done in vivo, and it was possible to demonstrate that the yeast GRASP fibrillates in starvation and heat-shock conditions. Here, we discuss the use of Fluorescence Lifetime Imaging Microscopy as a valid technique to help detect fibril formation in cell and also the possible implications of fibrillation for the formation of the Compartments for Unconventional Protein Secretion (CUPS). This work also contains initial experiments that point to a liquid-liquid phase separation of Grh1, an observation in consonance with the findings of intrinsic disorder and the recent proposition that the Golgi is actually an organelle phase separated from the cytosol. Finally, we present initial experiments of the characterization of Bug1, the golgin partner of Grh1. There are no structural data available on golgins in solution yet and their purification always presented an obstacle on obtaining them. We describe here a protocol capable of purifying Bug1 in high quantities, therefore paving the way for many other discoveries in the fields of protein secretion and liquid-liquid phase separation. GRASPs (de Golgi ReAssembly and Stacking Proteins) são proteínas inicialmente envolvidas na organização e manutenção do complexo de Golgi. Enquanto este papel vem sendo questionado nos últimos anos, muitas outras funções vem sendo a elas atribuídas. Em especial, destacamos a participação no ancoramento de vesículas de secreção que precisam atravessar o Golgi e processos de secreção não convencional. Estruturalmente, GRASPs podem ser divididas em um domínio N-terminal chamado GRASP e um domínio C-terminal que é altamente desordenado e não conservado, chamado SPR. Informação estrutural sobre GRASPs, até agora, tem sido muito escassa, o que motivou o início deste trabalho. Para estudar GRASPs, usamos a única GRASP da levedura Saccharomyces cereviseae (Grh1), um organismo modelo. Demonstramos que Grh1 possui regiões de desordem intrínseca também no domínio GRASP, sendo considerada uma proteína do tipo molten globule. Além disso, Grh1 é capaz de formar fibras do tipo amiloide quando em condições específicas in vitro, como baixo pH e temperatura levemente elevada. No objetivo de investigar uma possível correlação entre a formação de fibras e a função desempenhada por Grh1 em processos de secreção não convencional, parte desse trabalho foi realizada in vivo, e foi possível mostrar que a GRASP de levedura fibrila em condições específicas de privação de nutrientes e choque térmico. Aqui, discutimos o uso da microscopia do tempo de vida de fluorescência como uma técnica válida para auxiliar na detecção de formação de fibras in cell, e também as possíveis implicações da formação de fibras por GRASPs para formação de Compartimentos para Secreção Não Convencional (do inglês CUPS). Esse trabalho também contém experimentos iniciais que apontam para uma separação de fase líquido-líquido sofrida poe Grh1, o que estaria em consonância com os achados de desordem intrínseca e a recente proposta de que o Golgi seria, na verdade, uma organela em fase separada do citosol. No final deste trabalho, apresentamos ainda experimentos iniciais de caracterização de Bug1, a golgina parceira de Grh1. Não existem dados estruturais disponíveis para nenhuma golgina em solução, e sua purificação sempre se mostrou um obstáculo. Descrevemos aqui um protocolo através do qual foi possível se purificar Bug1 em grandes quantidades, abrindo caminho assim para que muitas outras descobertas sejam feitas no que diz respeito à secreção de proteínas e separação de fase líquido-líquido.