Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol−1, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol−1 and Tm raised by 5.6°C is of potential interest for trials in vivo.

Expression, stability, and membrane integration of truncation mutants of bovine rhodopsin

Premature termination of protein synthesis by nonsense mutations is at the molecular origin of a number of inherited disorders in the family of G protein-coupled seven-helix receptor proteins. To understand how such truncated polypeptides are processed by the cell, we have carried out COS-1 cell expression studies of mutants of bovine rhodopsin truncated at the first 1, 1.5, 2, 3, or 5 transmembrane segments (TMS) of the seven present in wild-type opsin. Our experiments show that successful completion of different stages in the cellular processing of the protein [membrane insertion, N-linked glycosylation, stability to proteolytic degradation, and transport from the endoplasmic reticulum (ER) membrane] requires progressively longer lengths of the polypeptide chain. Thus, none of the truncations affected the ability of the polypeptides to be integral membrane proteins. C-terminal truncations that generated polypeptides with fewer than two TMS resulted in misorientation and prevented glycosylation at the N terminus, whereas truncations that generated polypeptides with fewer than five TMS greatly destabilized the protein. However, all of the truncations prevented exit of the polypeptide from the ER. We conclude that during the biogenesis of rhodopsin, proper integration into the ER membrane occurs only after the synthesis of at least two TMS is completed. Synthesis of the next three TMS confers a gradual increase in stability, whereas the presence of more than five TMS is necessary for exit from the ER.

“Coarse” stability and bifurcation analysis using time-steppers: A reaction-diffusion example

Evolutionary, pattern forming partial differential equations (PDEs) are often derived as limiting descriptions of microscopic, kinetic theory-based models of molecular processes (e.g., reaction and diffusion). The PDE dynamic behavior can be probed through direct simulation (time integration) or, more systematically, through stability/bifurcation calculations; time-stepper-based approaches, like the Recursive Projection Method [Shroff, G. M. & Keller, H. B. (1993) SIAM J. Numer. Anal. 30, 1099–1120] provide an attractive framework for the latter. We demonstrate an adaptation of this approach that allows for a direct, effective (“coarse”) bifurcation analysis of microscopic, kinetic-based models; this is illustrated through a comparative study of the FitzHugh-Nagumo PDE and of a corresponding Lattice–Boltzmann model.

The MITE family Heartbreaker (Hbr): Molecular markers in maize

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 × Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for Mycobacterium tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the M. tuberculosis genome. MIRU-VNTR profiles of 72 different M. tuberculosis isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIRU-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all M. tuberculosis isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of M. tuberculosis.

Symmetry, stability, and dynamics of multidomain and multicomponent protein systems

Symmetry is commonly observed in many biological systems. Here we discuss representative examples of the role of symmetry in structural molecular biology. Point group symmetries are observed in many protein oligomers whose three-dimensional atomic structures have been elucidated by x-ray crystallography. Approximate symmetry also occurs in multidomain proteins. Symmetry often confers stability on the molecular system and results in economical usage of basic components to build the macromolecular structure. Symmetry is also associated with cooperativity. Mild perturbation from perfect symmetry may be essential in some systems for dynamic functions.

Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen1

Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativum L.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10−3 s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2Azz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2Azz measurements can provide an estimate of the optimum storage conditions.

Unraveling principles of lead discovery: from unfrustrated energy landscapes to novel molecular anchors.

The search for novel leads is a critical step in the drug discovery process. Computational approaches to identify new lead molecules have focused on discovering complete ligands by evaluating the binding affinity of a large number of candidates, a task of considerable complexity. A new computational method is introduced in this work based on the premise that the primary molecular recognition event in the protein binding site may be accomplished by small core fragments that serve as molecular anchors, providing a structurally stable platform that can be subsequently tailored into complete ligands. To fulfill its role, we show that an effective molecular anchor must meet both the thermodynamic requirement of relative energetic stability of a single binding mode and its consistent kinetic accessibility, which may be measured by the structural consensus of multiple docking simulations. From a large number of candidates, this technique is able to identify known core fragments responsible for primary recognition by the FK506 binding protein (FKBP-12), along with a diverse repertoire of novel molecular cores. By contrast, absolute energetic criteria for selecting molecular anchors are found to be promiscuous. A relationship between a minimum frustration principle of binding energy landscapes and receptor-specific molecular anchors in their role as "recognition nuclei" is established, thereby unraveling a mechanism of lead discovery and providing a practical route to receptor-biased computational combinatorial chemistry.

Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

Molecular basis for dysfunction of some mutant forms of methylmalonyl-CoA mutase: deductions from the structure of methionine synthase.

Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase. This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein. Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin. Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin. The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.

QSAR e dinâmica molecular no estudo de sistemas biomoleculares: predição da atividade biológica de antagonistas do receptor sigma-1 e simulações de bicamadas lipídicas

Diferentes abordagens teóricas têm sido utilizadas em estudos de sistemas biomoleculares com o objetivo de contribuir com o tratamento de diversas doenças. Para a dor neuropática, por exemplo, o estudo de compostos que interagem com o receptor sigma-1 (Sig-1R) pode elucidar os principais fatores associados à atividade biológica dos mesmos. Nesse propósito, estudos de Relações Quantitativas Estrutura-Atividade (QSAR) utilizando os métodos de regressão por Mínimos Quadrados Parciais (PLS) e Rede Neural Artificial (ANN) foram aplicados a 64 antagonistas do Sig-1R pertencentes à classe de 1-arilpirazóis. Modelos PLS e ANN foram utilizados com o objetivo de descrever comportamentos lineares e não lineares, respectivamente, entre um conjunto de descritores e a atividade biológica dos compostos selecionados. O modelo PLS foi obtido com 51 compostos no conjunto treinamento e 13 compostos no conjunto teste (r² = 0,768, q² = 0,684 e r²teste = 0,785). Testes de leave-N-out, randomização da atividade biológica e detecção de outliers confirmaram a robustez e estabilidade dos modelos e mostraram que os mesmos não foram obtidos por correlações ao acaso. Modelos também foram gerados a partir da Rede Neural Artificial Perceptron de Multicamadas (MLP-ANN), sendo que a arquitetura 6-12-1, treinada com as funções de transferência tansig-tansig, apresentou a melhor resposta para a predição da atividade biológica dos compostos (r²treinamento = 0,891, r²validação = 0,852 e r²teste = 0,793). Outra abordagem foi utilizada para simular o ambiente de membranas sinápticas utilizando bicamadas lipídicas compostas por POPC, DOPE, POPS e colesterol. Os estudos de dinâmica molecular desenvolvidos mostraram que altas concentrações de colesterol induzem redução da área por lipídeo e difusão lateral e aumento na espessura da membrana e nos valores de parâmetro de ordem causados pelo ordenamento das cadeias acil dos fosfolipídeos. As bicamadas lipídicas obtidas podem ser usadas para simular interações entre lipídeos e pequenas moléculas ou proteínas contribuindo para as pesquisas associadas a doenças como Alzheimer e Parkinson. As abordagens usadas nessa tese são essenciais para o desenvolvimento de novas pesquisas em Química Medicinal Computacional.

Clonagem molecular, expressão, purificação e caracterização estrutural da endoglucanase de Trichoderma harzianum visando o desenvolvimento de coquetéis enzimáticos para a produção de etanol lignocelulósico

O aumento na demanda mundial por energia, a perspectiva de encolhimento dos recursos energéticos e a preocupação global com a questão ambiental, despertaram o interesse por fontes alternativas de energia. A biomassa lignocelulósica é abundante e de baixo custo, com potencial para complementar a produção em larga escala de combustíveis. A degradação das moléculas constituintes da parede celular à açúcares fermentescíveis e então à etanol, ocorre através da hidrólise enzimática da biomassa. Contudo, a utilização de enzimas para esse fim encontra-se em estágio exploratório e representa um gargalo na implementação de tecnologias de etanol 2G em escala industrial, desencadeando a busca de celulases bioquimicamente mais ativas, estáveis e economicamente viáveis. O presente trabalho visou a caracterização da endoglucanase I do fungo Trichoderma harzianum, e para isso foi realizada expressão, ensaios bioquímicos e biofísicos do domínio catalítico (ThCel7B-CCD) e da proteína inteira (ThCel7B-full). A enzima exibiu um perfil acidofílico, com atividade ótima em pH 3,0 a 55°C. A proteína também se mostrou capaz de hidrolisar uma variedade de substratos, sendo a maior atividade hidrolítica em β-glucano (75 U mg-1). Ao analisar a estabilidade térmica medida a 55°C em pH 5, a atividade residual manteve-se intacta por mais de 2 meses. Outra característica relevante foi o elevado grau de sinergismo entre ThCel7B e ThCel7A. Análises de microscopia eletrônica de flocos de aveia submetidas à hidrólise com ThCel7B evidenciaram os efeitos de degradação do substrato em relação às amostras controle. O conjunto desses resultados, além de importante para a compreensão do mecanismo molecular de ThCel7B e de outras endoglucanases da família GH7, também revelou uma enzima de interesse biotecnológicos uma vez que o comportamento ácido e sua estabilidade térmica são características relevantes para aplicações industriais sob condições extremamente ácidas.

Novel silica membrane material for molecular sieve applications

Development of new silica membranes properties, e.g., molecular sieving properties, has been increasingly gaining importance in the last few years. A novel unsupported silica membrane, referred to as hydrophobic metal-doped silica, was developed by cobalt-doping within the organic templated silica matrix. The novel material was prepared by the acid-catalyzed hydrolysis and condensation process of tetraethylorthosilicate (TEOS) and methyltriethoxysilane (MTES), which is the precursor for methyl ligand covalently bounded to the silica matrix. The synthesis and surface properties of the novel unsupported silica membrane as well as the unsupported blank silica and modified silica membranes were revealed by surface and microstructural techniques, such as water contact angle measurement, FTIR, X-ray, Solid-state 29Si MAS NMR, TGA and N2 and CO2 adsorption measurements. The results showed that the thermal stability of the organic templated silica matrix was enhanced by cobalt-doping process. A hydrophobic microporous silica membrane material with high thermal stability up to ∼560 °C in oxidizing atmosphere and a narrow pore size distribution centered at 1.1 nm was obtained. Therefore, a novel precursor material for molecular sieve silica membranes applications has been achieved and developed.

X-Ray Imaging of SAPO-34 Molecular Sieves at the Nanoscale: Influence of Steaming on the Methanol-to-Hydrocarbons Reaction

The effect of a severe steaming treatment on the physicochemical properties and catalytic performance of H-SAPO-34 molecular sieves during the methanol-to-hydrocarbons (MTH) reaction has been investigated with a combination of scanning transmission X-ray microscopy (STXM), catalytic testing, and bulk characterization techniques, including ammonia temperature programmed desorption and 27Al and 29Si magic angle spinning nuclear magnetic resonance. For this purpose, two samples, namely a calcined and a steamed H-SAPO-34 catalyst powder, have been compared. It has been found that calcined H-SAPO-34 displays a high selectivity towards light olefins, yet shows a poor stability as compared to a zeolite H-ZSM-5 catalyst. Moreover, in situ STXM at the carbon K-edge during the MTH reaction allows construction of nanoscale chemical maps of the hydrocarbon species formed within the H-SAPO-34 aggregates as a function of reaction time and steam post-treatment. It was found that there is an initial preferential formation of coke precursor species within the core of the H-SAPO-34 aggregates. For longer times on stream the formation of the coke precursor species is extended to the outer regions, progressively filling the entire H-SAPO-34 catalyst particle. In contrast, the hydrothermally treated H-SAPO-34 showed similar reaction selectivity, but decreased activity and catalyst stability with respect to its calcined counterpart. These variations in MTH performance are related to a faster and more homogeneous formation of coke precursor species filling up the entire steamed H-SAPO-34 catalyst particle. Finally, the chemical imaging capabilities of the STXM method at the Al and Si K-edge are illustrated by visualizing the silicon islands at the nanoscale before and after steaming H-SAPO-34.

Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides; indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.