Identification of protein oligomerization states by analysis of interface conservation

The discrimination of true oligomeric protein–protein contacts from nonspecific crystal contacts remains problematic. Criteria that have been used previously base the assignment of oligomeric state on consideration of the area of the interface and/or the results of scoring functions based on statistical potentials. Both techniques have a high success rate but fail in more than 10% of cases. More importantly, the oligomeric states of several proteins are incorrectly assigned by both methods. Here we test the hypothesis that true oligomeric contacts should be identifiable on the basis of an increased degree of conservation of the residues involved in the interface. By quantifying the degree of conservation of the interface and comparing it with that of the remainder of the protein surface, we develop a new criterion that provides a highly effective complement to existing methods.

BimD/SPO76 is at the interface of cell cycle progression, chromosome morphogenesis, and recombination

BIMD of Aspergillus nidulans belongs to a highly conserved protein family implicated, in filamentous fungi, in sister-chromatid cohesion and DNA repair. We show here that BIMD is chromosome associated at all stages, except from late prophase through anaphase, during mitosis and meiosis, and is involved in several aspects of both programs. First, bimD+ function must be executed during S through M. Second, in bimD6 germlings, mitotic nuclear divisions and overall cellular program occur more rapidly than in wild type. Thus, BIMD, an abundant chromosomal protein, is a negative regulator of normal cell cycle progression. Third, bimD6 reduces the level of mitotic interhomolog recombination but does not alter the ratio between crossover and noncrossover outcomes. Moreover, bimD6 is normal for intrachromosomal recombination. Therefore, BIMD is probably not involved in the enzymology of recombinational repair per se. Finally, during meiosis, staining of the Sordaria ortholog Spo76p delineates robust chromosomal axes, whereas BIMD stains all chromatin. SPO76 and bimD are functional homologs with respect to their roles in mitotic chromosome metabolism but not in meiosis. We propose that BIMD exerts its diverse influences on cell cycle progression as well as chromosome morphogenesis and recombination by modulating chromosome structure.

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

Packing at the protein-water interface.

We have determined the packing efficiency at the protein-water interface by calculating the volumes of atoms on the protein surface and nearby water molecules in 22 crystal structures. We find that an atom on the protein surface occupies, on average, a volume approximately 7% larger than an atom of equivalent chemical type in the protein core. In these calculations, larger volumes result from voids between atoms and thus imply a looser or less efficient packing. We further find that the volumes of individual atoms are not related to their chemical type but rather to their structural location. More exposed atoms have larger volumes. Moreover, the packing around atoms in locally concave, grooved regions of protein surfaces is looser than that around atoms in locally convex, ridge regions. This as a direct manifestation of surface curvature-dependent hydration. The net volume increase for atoms on the protein surface is compensated by volume decreases in water molecules near the surface. These waters occupy volumes smaller than those in the bulk solvent by up to 20%; the precise amount of this decrease is directly related to the extent of contact with the protein.

Interface between culturally based preferences and genetic preferences: female mate choice in Poecilia reticulata.

The relative contribution of genetic and socio-cultural factors in the shaping of behavior is of fundamental importance to biologists and social scientists, yet it has proven to be extremely difficult to study in a controlled, experimental fashion. Here I describe experiments that examined the strength of genetic and cultural (imitative) factors in determining female mate choice in the guppy, Poecilia reticulata. Female guppies from the Paria River in Trinidad have a genetic, heritable preference for the amount of orange body color possessed by males. Female guppies will, however, also copy (imitate) the mate choice of other females in that when two males are matched for orange color, an "observer" female will copy the mate choice of another ("model") female. Three treatments were undertaken in which males differed by an average of 12%, 24%, or 40% of the total orange body color. In all cases, observer females viewed a model female prefer the less colorful male. When males differed by 12% or 24%, observer females preferred the less colorful male and thus copied the mate choice of others, despite a strong heritable preference for orange body color in males. When males differed by 40% orange body color, however, observer females preferred the more colorful male and did not copy the mate choice of the other female. In this system, then, imitation can "override" genetic preferences when the difference between orange body color in males is small or moderate, but genetic factors block out imitation effects when the difference in orange body color in males is large. This experiment provides the first attempt to experimentally examine the relative strength of cultural and genetic preferences for a particular trait and suggests that these two factors moderate one another in shaping social behavior.

Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.

The roles of language processing in a spoken language interface.

This paper provides an overview of the colloquium's discussion session on natural language understanding, which followed presentations by M. Bates [Bates, M. (1995) Proc. Natl. Acad. Sci. USA 92, 9977-9982] and R. C. Moore [Moore, R. C. (1995) Proc. Natl. Acad. Sci. USA 92, 9983-9988]. The paper reviews the dual role of language processing in providing understanding of the spoken input and an additional source of constraint in the recognition process. To date, language processing has successfully provided understanding but has provided only limited (and computationally expensive) constraint. As a result, most current systems use a loosely coupled, unidirectional interface, such as N-best or a word network, with natural language constraints as a postprocess, to filter or resort the recognizer output. However, the level of discourse context provides significant constraint on what people can talk about and how things can be referred to; when the system becomes an active participant, it can influence this order. But sources of discourse constraint have not been extensively explored, in part because these effects can only be seen by studying systems in the context of their use in interactive problem solving. This paper argues that we need to study interactive systems to understand what kinds of applications are appropriate for the current state of technology and how the technology can move from the laboratory toward real applications.

Commercial applications of speech interface technology: an industry at the threshold.

Speech interface technology, which includes automatic speech recognition, synthetic speech, and natural language processing, is beginning to have a significant impact on business and personal computer use. Today, powerful and inexpensive microprocessors and improved algorithms are driving commercial applications in computer command, consumer, data entry, speech-to-text, telephone, and voice verification. Robust speaker-independent recognition systems for command and navigation in personal computers are now available; telephone-based transaction and database inquiry systems using both speech synthesis and recognition are coming into use. Large-vocabulary speech interface systems for document creation and read-aloud proofing are expanding beyond niche markets. Today's applications represent a small preview of a rich future for speech interface technology that will eventually replace keyboards with microphones and loud-speakers to give easy accessibility to increasingly intelligent machines.

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.

Molecular approaches to smart materials via interface recognition and conformationally switchable cavitands

In this thesis the molecular level design of functional materials and systems is reported. In the first part, tetraphosphonate cavitand (Tiiii) recognition properties towards amino acids are studied both in the solid state, through single crystal X-ray diffraction, and in solution, via NMR and ITC experiments. The complexation ability of these supramolecular receptors is then applied to the detection of biologically remarkable N-methylated amino acids and peptides using complex dynamic emulsions-based sensing platforms. In the second part, a general supramolecular approach for surface decoration with single-molecule magnets (SMMs) is presented. The self-assembly of SMMs is achieved through the formation of a multiple hydrogen bonds architecture (UPy-NaPy complexation). Finally we explore the possibility to impart auxetic behavior to polymeric material through the introduction of conformationally switchable monomers, namely tetraquinoxaline cavitands (QxCav). Their interconversion from a closed vase conformation to an extended kite form is studied first in solution, then in polymeric matrixes via pH and tensile stimuli by UV-Vis spectroscopy.

Estudo fenomenológico e numérico do escoamento estratificado óleo-água ondulado e com mistura na interface

Escoamentos bifásicos estão presentes em diversos processos naturais e industriais, como na indústria de petróleo. Podem apresentar-se em diferentes configurações topológicas, ou, padrões de escoamento, entre eles o escoamento estratificado ondulado e o estratificado com mistura na interface. Os escoamentos bifásicos estratificados óleo-água têm sido utilizados como uma forma conveniente de evitar a formação de emulsões de água em óleo em oleodutos e possuem uma ocorrência comum em poços de petróleo direcionais. Quando a onda interfacial ultrapassa determinado limite geométrico e cinemático, surge o fenômeno do entranhamento de gotas, representado por misturas entre as fases junto à interface que promovem um aumento na queda de pressão. Modelos têm sido apresentados pela literatura na tentativa de descrever o fenômeno do entranhamento de gotas. Neste trabalho é apresentada uma nova proposta de modelagem matemática unidimensional para o entranhamento de gotas com o objetivo de melhorar a previsão dos parâmetros envolvidos, em especial, da fração volumétrica de óleo e da queda de pressão bifásica. Também foi utilizada simulação numérica computacional, CFD (Computational Fluid Dynamics), com o uso de software comercial para obtenção dos valores dos parâmetros do escoamento estratificado ondulado óleo-água (fração volumétrica de óleo, queda de pressão, amplitude e comprimento da onda interfacial). Os resultados da modelagem fenomenológica para entranhamento e os de CFD foram comparados com bancos de dados experimentais. Os resultados em CFD mostram concordância com os resultados experimentais, tanto na análise qualitativa das propriedades geométricas das ondas interfaciais, quanto na comparação direta com os dados para fração volumétrica e queda de pressão. Os resultados numéricos da modelagem fenomenológica para fatores de entranhamento apresentam boa concordância com dados da literatura.

Vídeo-Mapa: uma interface de geovisualização multimídia aplicada a transportes e meio ambiente.

A presente tese propõe uma metodologia de vídeo-mapeamento móvel georreferenciado a partir do desenvolvimento de protótipos que utilizam uma Interface de Geovisualização Multimídia para sincronizar o registro (em vídeo) de um local ou evento de interesse com a rota percorrida pelo veículo de inspeção (sobre mapa ou imagem), através da coleta de dados por sensores móveis: câmera digital, microfone, receptor GNSS e bússola digital. A interface permite a integração desses sensores com os atuais serviços de mapas digitais disponíveis na web. Sistemas como esse melhoram significativamente as análises temporais, a gestão e a tomada de decisão. A interface proposta e desenvolvida no presente trabalho é útil para muitas aplicações como ferramenta de monitoramento e inventário. Esta interface pode ser entendida como o componente visual de um sistema de mapeamento móvel ou como um sistema cartográfico alternativo ou complementar, para aplicações em que a precisão geométrica do receptor GNSS, na modalidade de navegação, é suficiente e sua acessibilidade, um fator competitivo. As aplicações desenvolvidas no presente trabalho foram duas: um sistema de monitoramento e inventário de placas de sinalização viária e um sistema de monitoramento de cheias/secas e inventário de propriedades na borda de reservatórios de hidroelétricas, ambos em pleno funcionamento.

Dependência de exercício físico em academias de ginástica e sua interface com suplementação e imagem corporal

O Brasil é o segundo país no mundo em número de academias de ginástica, contando atualmente com cerca de 30 mil unidades. Apesar da transição no modelo de gestão e filosofia das academias do fitness para o wellness, muitas ainda propagam em seus ambientes o culto ao corpo magro e atlético, dietas sem fundamentação nutricional e consumo descontrolado de suplementos. Crescem as evidências de que uma parcela significativa dos frequentadores de academias desenvolve um comprometimento exacerbado à prática de exercícios físicos, gerando até uma dependência patológica. O objetivo foi determinar a associação entre dependência de exercício físico (DEF), consumo de suplementos e insatisfação da imagem corporal em frequentadores de academias. Para tanto foi empregada a Escala de Comprometimento com Exercício Físico (CEF) em uma amostra de 227 frequentadores, maiores de 19 anos, de ambos os sexo em três academias. Foi realizada entrevista individual com emprego de anamnese nutricional para determinar o perfil de consumo suplementar, avaliação antropométrica, da imagem corporal, do compromisso ao exercício físico assim como de participação na academia. A frequência de DEF nas academias foi de 66% e sendo que as chances estimadas deste ser consumidor de suplementos de 4,53. Os indivíduos dependentes de exercício físico são jovens, com maior chance de pertencer ao sexo masculino, frequentar a academia > 5 vezes por semana, consumir mais de um tipo de suplemento e praticar musculação. A frequência de insatisfação com a imagem corporal foi alta tanto entre homens (50,9%) como mulheres (78,4%).