SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii.

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here we used small angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-ß-D-maltoside (DDM) fractionates on size exclusion chromatography (SEC) as two fractions. We show that in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an Alphafold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicates an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the MmIAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP to a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Simple Scattering: Lipid nanoparticle structural data repository.

Lipid nanoparticles (LNPs) are being intensively researched and developed to leverage their ability to safely and effectively deliver therapeutics. To achieve optimal therapeutic delivery, a comprehensive understanding of the relationship between formulation, structure, and efficacy is critical. However, the vast chemical space involved in the production of LNPs and the resulting structural complexity make the structure to function relationship challenging to assess and predict. New components and formulation procedures, which provide new opportunities for the use of LNPs, would be best identified and optimized using high-throughput characterization methods. Recently, a high-throughput workflow, consisting of automated mixing, small-angle X-ray scattering (SAXS), and cellular assays, demonstrated a link between formulation, internal structure, and efficacy for a library of LNPs. As SAXS data can be rapidly collected, the stage is set for the collection of thousands of SAXS profiles from a myriad of LNP formulations. In addition, correlated LNP small-angle neutron scattering (SANS) datasets, where components are systematically deuterated for additional contrast inside, provide complementary structural information. The centralization of SAXS and SANS datasets from LNPs, with appropriate, standardized metadata describing formulation parameters, into a data repository will provide valuable guidance for the formulation of LNPs with desired properties. To this end, we introduce Simple Scattering, an easy-to-use, open data repository for storing and sharing groups of correlated scattering profiles obtained from LNP screening experiments. Here, we discuss the current state of the repository, including limitations and upcoming changes, and our vision towards future usage in developing our collective knowledge base of LNPs.

Morphological Characterization of Self-Amplifying mRNA Lipid Nanoparticles.

The mRNA technology has emerged as a rapid modality to develop vaccines during pandemic situations with the potential to protect against endemic diseases. The success of mRNA in producing an antigen is dependent on the ability to deliver mRNA to the cells using a vehicle, which typically consists of a lipid nanoparticle (LNP). Self-amplifying mRNA (SAM) is a synthetic mRNA platform that, besides encoding for the antigen of interest, includes the replication machinery for mRNA amplification in the cells. Thus, SAM can generate many antigen encoding mRNA copies and prolong expression of the antigen with lower doses than those required for conventional mRNA. This work describes the morphology of LNPs containing encapsulated SAM (SAM LNPs), with SAM being three to four times larger than conventional mRNA. We show evidence that SAM changes its conformational structure when encapsulated in LNPs, becoming more compact than the free SAM form. A characteristic "bleb" structure is observed in SAM LNPs, which consists of a lipid-rich core and an aqueous RNA-rich core, both surrounded by a DSPC-rich lipid shell. We used SANS and SAXS data to confirm that the prevalent morphology of the LNP consists of two-core compartments where components are heterogeneously distributed between the two cores and the shell. A capped cylinder core-shell model with two interior compartments was built to capture the overall morphology of the LNP. These findings provide evidence that bleb two-compartment structures can be a representative morphology in SAM LNPs and highlight the need for additional studies that elucidate the role of spherical and bleb morphologies, their mechanisms of formation, and the parameters that lead to a particular morphology for a rational design of LNPs for mRNA delivery.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these are in turn determined by the multiscale structural features within condensates. However, structural characterizations of condensates are challenging, and hence rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and bespoke coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that mimic nucleolar granular components (GCs). We show that facsimiles of GCs are network fluids featuring spatial inhomogeneities across hierarchies of length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights, extracted from a combination of approaches, suggest that condensates formed by multivalent proteins share features with network fluids formed by associative systems such as patchy or hairy colloids.

SCOMAP-XD: atomistic deuterium contrast matching for small-angle neutron scattering in biology.

The contrast-variation method in small-angle neutron scattering (SANS) is a uniquely powerful technique for determining the structure of individual components in biomolecular systems containing regions of different neutron scattering length density ρ. By altering the ρ of the target solute and the solvent through judicious incorporation of deuterium, the scattering of desired solute features can be highlighted. Most contrast-variation methods focus on highlighting specific bulk solute elements, but not on how the scattering at specific scattering vectors q, which are associated with specific structural distances, changes with contrast. Indeed, many systems exhibit q-dependent contrast effects. Here, a method is presented for calculating both bulk contrast-match points and q-dependent contrast using 3D models with explicit solute and solvent atoms and SASSENA, an explicit-atom SANS calculator. The method calculates the bulk contrast-match points within 2.4% solvent D2O accuracy for test protein-nucleic acid and lipid nanodisc systems. The method incorporates a general model for the incorporation of deuterium at non-exchangeable sites that was derived by performing mass spectrometry on green fluorescent protein. The method also decomposes the scattering profile into its component parts and identifies structural features that change with contrast. The method is readily applicable to a variety of systems, will expand the understanding of q-dependent contrast matching and will aid in the optimization of next-generation neutron scattering experiments.

Change in Morphology of Dimyristoylphosphatidylcholine/Bile Salt Derivative Bicelle Assemblies with Dodecylmaltoside in the Disk and Ribbon Phases.

Bicelles, composed of a mixture of long and short chain lipids, form nanostructured molecular assemblies that are attractive lipid-membrane mimics for in vitro studies of integral membrane proteins. Here we study the effect of a third component, the single chain detergent n-dodecyl-ß-d-maltoside (DDM) on the morphology of bicelles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) below (10 °C) and above (38 °C) the phase transition. In the absence of DDM, bicelles convert from ellipsoidal disks at 10 °C to extended ribbon-like structures at 38 °C. The addition of DDM reshapes the ellipsoidal disc to a circular one and the flattened ribbon to a circular-cylinder worm-like micelle. Knowledge of the influence of the single chain detergent DDM on bicelle nanoscale morphology contributes toward comprehending lipid membrane self-organization and to the goal of optimizing lipid mimics for membrane biology research.

Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication.

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

Conformational Dynamics in the Interaction of SARS-CoV-2 Papain-like Protease with Human Interferon-Stimulated Gene 15 Protein.

Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques to study the mechanism of interaction of SARS-CoV-2 PLpro with hISG15. We showed that hISG15 undergoes a transition from an extended to a compact state after binding to PLpro, a conformation that has not been previously observed in complexes of SARS-CoV-2 PLpro with ISG15 from other species. Furthermore, computational analysis showed significant conformational flexibility in the ISG15 N-terminal domain, suggesting that it is weakly bound to PLpro and supports a binding mechanism that is dominated by the C-terminal ISG15 domain. This study fundamentally improves our understanding of the SARS-CoV-2 deISGylation complex that will help guide development of COVID-19 therapeutics targeting this complex.

Biosynthesis and characterization of deuterated chitosan in filamentous fungus and yeast.

Deuterated chitosan was produced from the filamentous fungus Rhizopus oryzae, cultivated with deuterated glucose in H2O medium, without the need for conventional chemical deacetylation. After extraction and purification, the chemical composition and structure were determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and small-angle neutron scattering (SANS). 13C NMR experiments provided additional information about the position of the deuterons in the glucoseamine backbone. The NMR spectra indicated that the deuterium incorporation at the non-exchangeable hydrogen positions of the aminoglucopyranosyl ring in the C3 - C5 positions was at least 60-80 %. However, the C2 position was deuterated at a much lower level (6%). Also, SANS showed that the structure of deuterated chitosan was very similar compared to the non-deuterated counterpart. The most abundant radii of the protiated and deuterated chitosan fibers were 54 Å and 60 Å, respectively, but there is a broader distribution of fiber radii in the protiated chitosan sample. The highly deuterated, soluble fungal chitosan described here can be used as a model material for studying chitosan-enzyme complexes for future neutron scattering studies. Because the physical behavior of non-deuterated fungal chitosan mimicked that of shrimp shell chitosan, the methods presented here represent a new approach to producing a high quality deuterated non-animal-derived aminopolysaccharide for studying the structure-function association of biocomposite materials in drug delivery, tissue engineering and other bioactive chitosan-based composites.

MAW point mutation impairs H. Seropedicae RecA ATP hydrolysis and DNA repair without inducing large conformational changes in its structure.

RecA is a multifunctional protein that plays a central role in DNA repair in bacteria. The structural Make ATP Work motif (MAW) is proposed to control the ATPase activity of RecA. In the present work, we report the biochemical activity and structural effects of the L53Q mutation at the MAW motif of the RecA protein from H. seropedicae (HsRecA L53Q). In vitro studies showed that HsRecA L53Q can bind ADP, ATP, and ssDNA, as does wild-type RecA. However, the ATPase and DNA-strand exchange activities were completely lost. In vivo studies showed that the expression of HsRecA L53Q in E. coli recA1 does not change its phenotype when cells were challenged with MMS and UV. Molecular dynamics simulations showed the L53Q point mutation did not cause large conformational changes in the HsRecA structure. However, there is a difference on dynamical cross-correlation movements of the residues involved in contacts within the ATP binding site and regions that hold the DNA binding sites. Additionally, a new hydrogen bond, formed between Q53 and T49, was hypothesized to allow an independent motion of the MAW motif from the hydrophobic core, what could explain the observed loss of activity of HsRecA L53Q.

Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament.

The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

Tradução e adaptação cultural da barratt impulsiveness scale (BIS-11) para aplicação em adultos brasileiros / Translation and cultural adaptation of barratt impulsiveness scale (BIS-11) for administration in Brazilian adults

OBJETIVOS: Os objetivos deste estudo foram traduzir, adaptar culturalmente e verificar a equivalência literal, semântica e idiomática da Barratt Impulsiveness Scale (BIS-11), que avalia a presença de manifestações da impulsividade tendo como base o modelo teórico proposto por Ernst Barratt. MÉTODOS: Inicialmente, a versão original em inglês da BIS-11 foi traduzida para o português por seis pesquisadores bilíngues. Em seguida, foi realizada uma tradução reversa para o inglês por uma tradutora de origem norte-americana. As versões original, traduzida e retraduzida foram avaliadas por um comitê de juízes especialistas, os quais emitiram pareceres com as observações pertinentes, o que culminou em uma versão final traduzida da BIS-11. As versões original e traduzida foram aplicadas em duas amostras da população geral com proficiência na língua inglesa, a fim de investigar a equivalência literal, semântica e idiomática da versão traduzida por meio de análises de correlação. CONCLUSÃO: Os resultados das análises quantitativas indicaram que a versão final do instrumento é satisfatória.

OBJECTIVES: The objective of this study was to translate, make transcultural adaptation and assess the semantic, idiomatic and literal equivalence of the Barratt Impulsiveness Scale (BIS-11). METHODS:This scale assesses the presence of impulsive manifestations from the theoretical model proposed by Ernst Barratt. Firstly, the BIS-11 original version in English was translated to Portuguese by six bilingual researches. After this, was made the back-translation to English by a translator that was born in United States. Then, the three versions (original, translated and back-translated) were assessed by a specialists committee which made and analyze and comments about the process and then we reach the final translated version of BIS-11. The original and translated version of BIS-11 was applied in two samples from general population with proficiency both in English and Portuguese. This method was adopted to assess the literal, semantic e idiomatic equivalence of these versions by mean of correlation analyses. CONCLUSION: The final results of quantitative analyses show that the final version of BIS-11 is satisfactory.

Brazilian Portuguese version of the Iowa Gambling Task: transcultural adaptation and discriminant validity / Versão em português do Iowa Gambling Test: adaptação transcultural e validade discriminate

OBJECTIVE: The Iowa Gambling Task is a neuropsychological task developed in English, most widely used to assess decision-making. The aim of this work was to adapt the Iowa Gambling Task to Brazilian Portuguese, compare it with the original version and assess its validity. METHOD: We assessed 75 Brazilian adults divided into three groups: 1) 25 healthy volunteers holding the Proficiency Certificate in English tested using the English version of the Iowa Gambling Task; 2) 25 healthy volunteers who did not speak or read English tested using the Iowa Gambling Task-Portuguese; 3) 25 Attention Deficit Hyperactivity Disorder subjects tested with the Iowa Gambling Task-Portuguese. RESULTS: No difference between groups 1 and 2 was observed. Nonetheless, we found significant differences between Attention Deficit Hyperactivity Disorder subjects and the other 2 groups on blocks 3, 4, 5, and on net score. CONCLUSION: Our results are similar to those previously described in the literature concerning adults without neuropsychiatric diseases. Since those two versions were equivalent and Attention Deficit Hyperactivity Disorder subjects performed significantly worse than healthy volunteers we can conclude that the adaptation of the Iowa Gambling Task to Brazilian Portuguese is valid and can be used for research purposes in the Brazilian context.

OBJETIVO: Iowa Gambling Task é uma tarefa neuropsicológica originalmente desenvolvida em inglês, mais usada no mundo para avaliar o processo de tomada de decisões. Este estudo pretendeu adaptar o Iowa Gambling Task para o português, comparar a versão adaptada com a versão original em inglês e avaliar sua validade discriminante. MÉTODO: Foram investigados 75 adultos brasileiros divididos em três grupos: 1) 25 voluntários sadios proficientes em inglês, avaliados com a versão original em inglês; 2) 25 voluntários sadios não-proficientes em inglês avaliados com o Iowa Gambling Task-português; 3) 25 adultos com Transtorno do Déficit de Atenção e Hiperatividade (avaliados com o Iowa Gambling Task-português. RESULTADOS: Não houve diferenças entre os grupos 1 e 2. No entanto, encontramos diferenças entre os adultos com Transtorno do Déficit de Atenção e Hiperatividade e os outros dois grupos nos blocos 3, 4, 5 e no netscore. CONCLUSÃO: Nossos resultados são semelhantes aos descritos na literatura. Considerando que as duas versões se mostraram equivalentes e os sujeitos com Transtorno do Déficit de Atenção e Hiperatividade desempenharam significativamente pior do que os controles, podemos concluir que a adaptação do Iowa Gambling Task para o português praticado no Brasil é válida e pode ser aplicada no contexto brasileiro.

Brazilian Portuguese version of the Iowa Gambling Task: transcultural adaptation and discriminant validity.

OBJECTIVE: The Iowa Gambling Task is a neuropsychological task developed in English, most widely used to assess decision-making. The aim of this work was to adapt the Iowa Gambling Task to Brazilian Portuguese, compare it with the original version and assess its validity. METHOD: We assessed 75 Brazilian adults divided into three groups: 1) 25 healthy volunteers holding the Proficiency Certificate in English tested using the English version of the Iowa Gambling Task; 2) 25 healthy volunteers who did not speak or read English tested using the Iowa Gambling Task-Portuguese; 3) 25 Attention Deficit Hyperactivity Disorder subjects tested with the Iowa Gambling Task-Portuguese. RESULTS: No difference between groups 1 and 2 was observed. Nonetheless, we found significant differences between Attention Deficit Hyperactivity Disorder subjects and the other 2 groups on blocks 3, 4, 5, and on net score. CONCLUSION: Our results are similar to those previously described in the literature concerning adults without neuropsychiatric diseases. Since those two versions were equivalent and Attention Deficit Hyperactivity Disorder subjects performed significantly worse than healthy volunteers we can conclude that the adaptation of the Iowa Gambling Task to Brazilian Portuguese is valid and can be used for research purposes in the Brazilian context.

Planning abilities of children aged 4 years and 9 months to 8 ½ years: Effects of age, fluid intelligence and school type on performance in the Tower of London test.

The present study investigated the relationship between age and one type of environmental factor, namely, type of school (i.e., private vs. public), and the development of mental planning ability, as measured by the Tower of London (TOL) test. METHODS: Participants comprised 197 public and 174 private school students, ranging in age from 4 years and 9 months to 8 years and 6 months. Besides the TOL test, students were administered Raven's Colored Matrices. RESULTS: Results confirmed the findings of previous studies that both age and school type are important predictors of mental planning. Furthermore, results also suggest that the relationship between type of school and mental planning ability cannot be accounted for by differences in students' fluid intelligence. CONCLUSION: In the present study, the TOL test continued to differentiate public from private school students, even after we controlled for the effect of differences on the Raven test.

O presente estudo investigou a relação entre idade, e um tipo de fator ambiental, o tipo de ambiente escolar (público x privado) e o desenvolvimento das habilidades mentais de planejamento medidas pelo teste da Torre de Londres (TOL). MÉTODOS: Os participantes foram 197 estudantes de escolas públicas e 174 de escolas privadas com idade variando de 4 anos e 9 meses a 8 anos e 6 meses. Além do TOL, foi administrado o teste das Matrizes Coloridas de Raven. RESULTADOS: Os resultados confirmam os achados de estudos prévios de que tanto a idade e o tipo de escola são importantes preditores das habilidades de planejamento. Além disso, eles também sugerem que a relação entre o tipo de escola e as habilidades de planejamento não podem ser explicadas por diferenças na inteligência fluída dos estudantes. CONCLUSÃO: No presente estudo, o teste da torre de Londres diferenciou crianças de escolas públicas e privadas mesmo após ter sido controlado o efeito das diferenças medidas pelo teste das Matrizes Coloridas de Raven.

Aterros sanitarios: análise da reprodutibilidade de parámetros que influenciam o processo da digestao anaeróbia

El presente trabajo se desenvolvió a partir de la construcción experimental de rellenos sanitarios de residuos sólidos domésticos, en escala real, que han permitido el análisis del comportamiento de parámetros que controlan la digestión anaerobia de los residuos. La preocupación del autor fué verificar, estadísticamente, cuales son los parámetros relevantes en la digestión de residuos que forman el relleno experimental, durante el período de análisis. El material utilizado en este estudio es la basura domiciliaria de la ciudad de Sao Carlos, Estado de Sao Paulo