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Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of flexibility and rigidity are fundamental for understanding the relationships between function, structure and stability. Protein unfolding can often be triggered by thermal fluctuations with flexible residues usually on the protein surface. Therefore, identification and knowledge of the effect of modification to flexible regions in protein structures are required for efficient protein engineering and the rational design of thermally stable proteins. The most flexible regions in protein are loops, hence their rigidification is one of the effective strategies for increasing thermal stability. Directed evolution or rational design by computational prediction can also lead to the generation of thermally stable proteins. Computational protein design has been improved significantly in recent years and has successfully produced de novo stable backbone structures with optimized sequences and functions. This review discusses intramolecular and intermolecular interactions that determine the protein structure, and the strategies utilized in the mutagenesis of mesophilic proteins to stabilize and improve the functional characteristics of biocatalysts by describing efficient techniques and strategies to rigidify flexible loops at appropriate positions in the structure of the protein.

This paper introduces the concept of “bypass urbanism” to account for a process of urbanization that is reordering center-periphery relations of urban regions into new hierarchies. Bypass urbanism became visible through a comparison of large-scale urban transformations at the peripheries of Kolkata, Lagos, and Mexico City by zooming out and considering their impacts on the socio-spatial structure of the extended urban regions. Bypass urbanism is not emerging from the construction of a singular new town or real estate project, but is the result of the simultaneous development of an ensemble of various independent but related projects. Therefore, bypass urbanism usually does not emanate from a coherent planning initiative, even less so from a hidden “master plan” at the hands of any single developer or state agency, but it emerges through a convergence of interests over large areas of land at the geographical periphery of urban regions that have been made available for new urban developments by various measures. We understand bypass urbanism as a multidimensional process that includes material-geographical bypassing, the bypassing of regulatory frameworks, and socio-economic bypassing in everyday life. It results in the creation of exclusive and excluding spaces that enable middle and upper-class lifestyles, at the same time leading to the peripheralization of extant urban areas that are bypassed and neglected. The massive scale of bypass urbanism that we have observed represents a new quality of urban development resulting not in isolated urban enclaves or archipelagos, but in the fundamental restructuring of the extended urban region with far reaching and incalculable repercussions., Environment and Planning A: Economy and Space, 53 (4), ISSN:0308-518X, ISSN:1472-3409

International audience; This article introduces the concept of popular urbanization to describe a specific urbanization process based on collective initiatives, self‐organization and the activities of inhabitants. We understand popular urbanization as an urban strategy through which an urban territory is produced, transformed and appropriated by the people. This concept results from a theoretically guided and empirically grounded comparison of Mexico City, Istanbul and Lagos. Based on postcolonial critiques of urban theory and on the epistemologies of planetary urbanization, we bring urbanization processes in these urban regions into conversation with each other through a multidimensional theoretical framework inspired by Henri Lefebvre focusing on material interaction, territorial regulation, and everyday experience. In this way, popular urbanization emerged as a distinct urbanization process, which we identified in all three contexts. While this process is often subsumed under the broader concept of ‘urban informality’, we suggest that it may be helpful to distinguish popular urbanization as primarily led by the people, while commodification and state agencies play minor roles. As popular urbanization unfolds in diverse ways dependent upon the wider urban context, specific political constellations and actions, it results in a variety of spatial outcomes and temporal trajectories. This is therefore a revisable and open concept. In proposing the concept of popular urbanization for further examination, we seek to contribute to the collective development of a decentered vocabulary of urbanization.

International audience; The numerous advantages that nanotechnology can bring to the design of pharmaceutical delivery systems are perhaps nowhere better illustrated than in the area of cancer treatment. These nanostructures not only can improve the anti-proliferative properties of the drugs but also ameliorate the side effects by providing site-specific drug delivery. Furthermore, food-based proteins, due to their biocompatibility and biodegradability, provide a ready-made ingredient as a carrier for various chemotherapeutic agents. The most popular proteins in this field are milk proteins including beta-lactoglobulin (beta-LG), lactoferrin (LF), and the caseins (CN), all of which have desirable properties to make them ideal for this purpose. Grafting these two approaches, different milk protein-based nanodrugs have been synthesized that show promising potential in providing more targeted delivery of drugs thereby reducing the cytotoxic effects of chemotherapy. In some cases, being encapsulated in these proteins (in nanoscale), the chemotherapeutic agents can be consumed orally while enhancing site-specific delivery. This article reviews various nanodrug delivery systems containing milk proteins, which are designed for cancer treatment. Moreover, their physical and their anti-proliferative characteristics are briefly described. Graphic abstract

This special issue on the role of traditional authorities in African cities highlights critical debates about governance and urban development on a fast-urbanising continent. The six articles in this issue focus on the following: (1) the roles of traditional authorities as custodians of the values of society; (2) the roles of traditional leaders as moral authorities; (3) the modern chieftaincy as an invention of the colonial state; (4) the ‘unrelenting co-optation and appropriation’ of traditional governance structures by the state; and (5) the stretching of pre-colonial narratives to justify the legitimacy of traditional leadership and its control of community resources. The special issue features contributions from Burkina Faso, Senegal, Ghana, South Africa, Botswana and Eswatini, providing a rare comparison between cases from Southern and West Africa.

International audience; With this paper, we analyse an ordinary urban process, which has received little attention so far, and propose a new concept to take account of it: plotting urbanism. It is usually subsumed under terms like “urban informality” or “incremental urbanism” and not studied as a distinct process. In comparing Lagos, Istanbul and Shenzhen we captured four defining features of plotting urbanism: first, it unfolds in a piecemeal fashion with limited comprehensive planning. Second, it emerges from specific territorial compromises often resulting from conflicts between overlapping modes of territorial regulation, land tenure and property rights. Third, plotting is based on commodification of housing and land, which might accentuate socio‐economic differentiations between property‐owners, who often live in the same area, and their tenants. The term “plotting” highlights the key role of the plot in the process. It also alludes to strategic acts of collaboration for individual and collective benefit.

Among the various methods used to induce protein amyloid aggregation in vitro (quiescent/agitated/shaken incubation) and different strategies of modulating the aggregation, the quiescent incubation of an amyloidogenic protein (AP) solution at a defined temperature, and using polyphenolic small molecules, are most common. Using a typical ThT fluorescence assay, we have shown that at the lowest percentage of DMSO used, the thermomixer-shaken incubation of AP solution results in a high-yielding aggregation. Under these conditions, some polyphenols such as curcumin and quercetin are unable to redirect the process except at higher concentrations, as shown by CD spectroscopy and TEM. Based upon NMR experiments and MD simulations, we suggest that polyphenolic self-association is the main cause. Because of the different properties of n-mer particles of these polyphenols formed through self-association, the redirection of AP aggregation occurred differently. To prove the involvement of self-association, rosmarinic acid with a much lower tendency to self-associate was examined. It was unable to redirect the process even at concentrations two and three times those of the other two.

International audience; Contemporary processes of urbanisation present major challenges for urban research and theory as urban areas expand and interweave. In this process, urban forms are constantly changing and new urban configurations are frequently evolving. An adequate understanding of urbanisation must derive its empirical and theoretical inspirations from the multitude of urban experiences across the various divides that shape the contemporary world. New concepts and terms are urgently required that would help, both analytically and cartographically, to decipher the differentiated and rapidly mutating landscapes of urbanisation that are being produced today. One of the key procedures to address these challenges is the application of comparative strategies. Based on postcolonial critiques of urban theory and on the epistemologies of planetary urbanisation, this paper introduces and discusses the theoretical and methodological framework of a collaborative comparative study of urbanisation processes in eight large metropolitan territories across the world: Tokyo, Hong Kong/Shenzhen/Dongguan, Kolkata, Istanbul, Lagos, Paris, Mexico City and Los Angeles. In order to approach these large territories, a specific methodological design is applied mainly based on qualitative methods and a newly developed method of mapping. After thepresentation of the main lines of our theoretical and methodological approach we discuss some of the new comparative concepts that we developed through this process: popular urbanisation, plotting urbanism, multilayered patchwork urbanisation and the incorporation of urban differences.; 随着城市地区的扩大和相互交织，当代城市化进程给城市研究和理论带来了重大挑战。在这个过程中，城市形态不断变化，新的城市形态在不断演进。要充分理解城市化，我们必须从塑造当代世界的大量纷纭歧出的城市经验中获得经验和理论层面的启发。如今正在产生的城市化格局千差万别，并迅速变化，我们迫切需要新的概念和术语，在分析层面和图解层面帮助破译这些格局。应对这些挑战的关键手段是应用比较策略。本文基于城市理论的后殖民批判和全域城市化的认识论，介绍并讨论了对世界八大都市圈城市化进程进行协同比较研究的理论和方法论框架，这八大都市圈是：东京、香港/深圳/东莞、加尔各答、伊斯坦布尔、拉各斯、巴黎、墨西哥城和洛杉矶。为着手探讨这些广大的都市圈，我们主要基于定性方法和新开发的测绘方法，应用了一套特定的方法论设计。在介绍了我们主要的理论和方法论进路之后，我们讨论了在这个过程中发展起来的一些新的比较概念：大众城市化、小块圈地式城市化、多层拼凑城市化以及城市差异的纳入。

Thanks to the progress of nanotechnology there are several agent-delivery systems that can be selected to achieve rapid and specific delivery of a wide variety of biologically active agents. Consequently, the manipulation and engineering of biopolymers has become one of the most exciting subjects for those who study delivery systems on the nanoscale. In this regard, both nanoparticle formation and a carrier role have been observed in the case of the globular milk whey protein, beta-lactoglobulin (beta-LG), setting it apart from many other proteins. To date, many efforts adopting different approaches have created beta-LG nanoparticles useful in forming delivery systems for various agents with specific targets. In this review, the potential of beta-LG to play the role of an efficient and diverse carrier protein, as well as its ability to form a well-targeted nano-scale delivery system is discussed.

International audience; The milk protein beta-lactoglobulin has been widely studied since its discovery, both as a purified protein and in mixtures with other milk proteins, where its effect on the processing properties is of importance to the dairy industry. The protein can bind a variety of small hydrophobic molecules, which may allow its use as an oral delivery vehicle. In the present study we have examined the binding of odd-numbered fatty acids by isothermal calorimetry (ITC), X-ray crystallography and computer modelling to provide a clearer picture of the extent and variability of the central binding pocket. The Kd values for the fatty acids C13, C15, C16, C17 and C19 as determined by ITC are 1.93, 2.91, 3.05, 4.11 and 8.67 x 10(-7) M, respectively. The molecular structures revealed the ligands bound in the central cavity with generally well ordered lipophilic tails but significant positional variation at the carboxyl group end. In silico docking analyses identified the lipophilic interactions within the central cavity as the main driving force for binding with electrostatic interactions and H-bonds playing a minor role.

The crystal structure of the triclinic form of the milk protein β-lactoglobulin from sheep (Ovis aries) at 1.1 Å resolution is described together with a comparison of the triclinic structures of the low-pH bovine and high-pH ovine proteins. All three structures are remarkably similar, despite the well known pH-dependent conformational transition described for the bovine and porcine proteins that occurs in solution. The high resolution of the present structure determination has allowed a more accurate description of the protein than has hitherto been possible, but it is still not clear whether flexibility changes in the external loops can compensate for the presence of a significant void in the unliganded interior of the structure.

The peripheral contexts of urban agglomerations in sub-Saharan Africa are usually discussed in reference to peri-urban literature, which tends to be largely descriptive rather than theoretically engaged. The urban peripheral contexts of North America and Europe, however, have been highly differentiated as part of suburban literature, with a flood of proposed terms to better address them. The goal of this article, whilst engaging with peri-urban literature, is to confront the urbanisation occurring at the peripheries of Lagos with suburban theory. For the peripheries of sub-Saharan Africa to contribute meaningfully to the suburban debate, they must first be differentiated. This article attempts to show the diversity of forms, actors and processes that are producing peripheral expansion in Lagos, and the complexity and ambiguity of the multiple land systems at work. This challenges any easy assumptions about pervasive poverty and informality in the urban peripheries of Lagos, much less sub-Saharan Africa in...

Colon cancer is one of the most common internal malignancies, and conventional chemotherapy is not effective in its treatment. Nanoparticles hold tremendous potential as an effective drug delivery system. The physicochemical properties of β-lactoglobulin, the main whey protein of cow's milk, such as its stability at low pH, its resistance to gastric protease, and its ability to bind hydrophobic ligands, give it potential for transporting drugs specifically for colon cancer. In the present research, β-lactoglobulin-pectin nanoparticles were designed to transfer a newly synthesized, anticancer platinum complex (bipyridine ethyl dithiocarbamate Pt(II) nitrate), to the colon. The effects of multiple factors on the size and the colloidal stability of the nanoparticles were studied using dynamic light scattering and scanning electron microscopy techniques. Results showed that the best particle size and highest colloidal stability were obtained in phosphate buffer, pH 4.5, with 0.5 mg/mL β-lactoglobulin and 0.025-0.05wt% pectin. The drug release profile in simulated gastrointestinal conditions demonstrated that β-lactoglobulin with a secondary coating is stable in acidic conditions but is able to release its cargo at pH 7. Hence, these nanoparticles have potential to serve as novel and effective vehicles for oral drug delivery preparations.

β-Lactoglobulin is a small globular protein found in ruminant milk and many, but not all, other mammals. The cow protein is the major whey protein synthesized as a 180-amino-acid polypeptide from which an 18-residue signal peptide is removed on secretion. Genetic variants involving a few amino acid positions have been reported for many species. The cow protein is stable between pH 3 and 7 but undergoes conformational change and unfolding as the pH is raised further. The structure is a conical barrel, open at one end, comprising eight antiparallel β-strands with a three-turn α-helix on the outer surface of the barrel. While the ruminant forms of the protein are dimeric, those from other species appear to be monomeric. The protein belongs to a widespread family of small, mostly secretory proteins called the lipocalins, since most members appear to carry small hydrophobic ligands internally. The function of the protein is unknown, although it is a good nutritional source of amino acids, and a transport role of some kind has been suggested. β-Lactoglobulin binds a wide variety of ligands and has been studied intensively not only as a model protein but also because it affects milk stability during commercial processing, its five cysteine residues allowing disulfide interchange to play a key role.

The deep-sea bacterium, Photobacterium profundum SS9, has been adopted as a model organism to understand the molecular basis of cold-adapted high-pressure-loving (piezophilic) growth. Despite growing optimally at 28 MPa (15° C), P. profundum SS9 can grow over a wide range of pressures and temperatures. The ability to grow at atmospheric pressure has enabled a limited set of genetic tools to be developed, which has provided genetic insights into the mechanism of piezophilic growth in P. profundum SS9. This review focuses on how genetic studies have uncovered the importance of processes affecting the DNA and the bacterial cell envelope in the piezophilic growth of P. profundum SS9. In addition, a method was developed to assess quantitative piezophilic colony growth of P. profundum SS9 on solid agar. Future studies, using this methodology, could provide novel insights into the molecular basis of piezophilic, surface-attached growth.

There are abundant examples of nanoclusters and inorganic microcrystals in biology. Their study under physiologically relevant conditions remains challenging due to their heterogeneity, instability, and the requirements of sample preparation. Advantages of using neutron diffraction and contrast matching to characterize biomaterials are highlighted in this article. We have applied these and complementary techniques to search for nanocrystals within clusters of calcium phosphate sequestered by bovine phosphopeptides, derived from osteopontin or casein. The neutron diffraction patterns show broad features that could be consistent with hexagonal hydroxyapatite crystallites smaller than 18.9 A. Such nanocrystallites are, however, undetected by the complementary X-ray and FTIR data, collected on the same samples. The absence of a distinct diffraction pattern from the nanoclusters supports the generally accepted amorphous calcium phosphate structure of the mineral core.

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.

β -Lactoglobulin ( β -LG) is the major whey protein of ruminant species and is also present in the milks of many, but not all, other species. Its amino-acid sequence and 3-dimensional structure show that it is a lipocalin, a widely diverse family, most of which bind small hydrophobic ligands and thus may act as specific transporters, as does serum retinol binding protein. Bovine β -LG binds a wide range of ligands, but this may not be the reason for its presence in milk. In reviewing the structure and physicochemical properties of the protein, we present the structures of the ligands cholesterol (at a resolution of 2.0A, R=0.221; R free = 0.295) and vitamin D 2 (at a resolution of 2.4A, R=0.212; R free = 0.297) each bound to the central binding cavity of bovine β -LG at pH 7.3. Neither ligand is fully visible in the electron density maps, and the less well-ordered regions are the polar end groups at the mouth of the binding site. In a separate experiment, a mercury ion was bound to the free Cys121 (at a resolution of 2.2A, R=0.218; R free = 0.288) in a way that transmitted a small structural change through Asp137 via Arg148 to the dimer interface. It is not clear if the known dissociation that arises from the reaction of β -LG with HgCl 2 results from this perturbation. In reviewing the structural studies that reveal the ligand binding sites for long-chain fatty acids, retinoids, and steroids, only the central location, common to all lipocalins so far examined, is occupied under the conditions used. We find that there is no crystallographic evidence of another ligand binding site in our crystals grown in approximately 1.3 M citrate, although low ionic strength studies in solution indicate the possible presence of at least one other low affinity site. The apparent ability of the binding site to accommodate a wide range of ligands may point to a possible physiological function. However, by considering the lipocalin family in general, and the species distribution of β -LG in particular, some speculation as to the physiological function can be made. β -Lactoglobulin has been reported as being implicated, inter alia, in hydrophobic ligand transport and uptake, enzyme regulation, and the neonatal acquisition of passive immunity. However, these functions do not appear to be consistent between species. Sequence comparisons among members of the lipocalin family reveal that glycodelin, found in the human endometrium during early pregnancy, is the most closely related to β -LG. Although the function of glycodelin is also unknown, it appears to have effects on the immune system and/or to be involved in differentiation. It is proposed that β -LG, over-expressed in the lactating mammary gland of many, but not all, species, is primarily an important source of amino acids for the offspring of those animals that produce it, but that this function arose by gene duplication from the physiologically essential glycodelin. The other functions that have been associated with β -LG in the neonate are, therefore, fortuitous.

5-Aminolevulinic acid synthase (ALAS), the first enzyme of the heme biosynthesis pathway, catalyses the pyridoxal 5'-phosphate-dependent condensation between glycine and succinyl-CoA to yield 5-aminolevulinic acid (5-amino-4-oxopentanoate). A three-dimensional structural model of Rhodobacter spheroides ALAS has been constructed and used to identify amino acid residues at the active site that are likely to be important for the recognition of glycine, the only amino acid substrate. Several residues have been investigated by site-directed mutagenesis and enzyme variants have been generated that are able to use alanine, serine or threonine. A three-dimensional structure model of 5-aminolevulinic acid synthase from human erythrocytes (ALAS 2) has also been constructed and used to map a range of naturally occurring human mutants that give rise to X-linked sideroblastic anemia. A number of these anemias respond favourably to vitamin B(6) (pyridoxine) therapy, whereas others are either partially responsive or completely refractory. Detailed investigations with selected human mutants have highlighted the importance of arginine-517 that is implicated in glycine carboxyl group binding.

This paper describes the possible usefulness of knowledge of the three-dimensional structure of milk proteins to dairy scientists. After a brief introduction of the available methodology and the structures of milk proteins that are already available, the structure of bovine β-lactoglobulin is used to illustrate the possible applications of the structure to understanding the problems to which the protein contributes during milk processing.

We measured in-situ the volumetric changes to the molecular structure of the bovine milk protein β-lactoglobulin in a series of high-pressure processing experiments. We used the so-called μPVT device at hydrostatic pressures up to 500 MPa under isothermal conditions of 25 °C, the protein in solution, prepared at both pH 5.0 and 7.0, in a series of compression and decompression cycles. Significant irreversible volumetric changes were found, most notably with a marked step decrease in molar volume of 1.3 litre mol-1 occurring between 10 and 17 MPa. Irreversible molar volume changes of 2.16 litre mol-1 were found when pressures above 17 MPa were applied. This is thought to be due in part to a collapse of the inner calyx. Volume changes were confirmed by measuring the unit-cell volume of the crystalline protein. The effects of pH were indistinguishable. These pressures are considerably less than previously thought necessary for this protein.

The crystal structures of beta-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C222(1) (lattice Y) by X-ray diffraction at 2.0 A and 1.95 A resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N.Jameson, G.B. (1999) Protein Sci. 8, 75-83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.

The lipocalin family became established shortly after the structural similarity was noted between plasma retinol binding protein and the bovine milk protein, β-lactoglobulin. During the past 60 years, β-lactoglobulin has been studied by essentially every biochemical technique available and so there is a huge literature upon its properties. Despite all of these studies, no specific biological function has been ascribed definitively to the protein, although several possibilities have been suggested. During the processing of milk on an industrial scale, the unpredictable nature of the process has been put down to the presence of β-lactoglobulin and certainly the whey protein has been implicated in the initiation of aggregation that leads to the fouling of heat exchangers. This short review of the properties of the protein will concentrate mainly on studies carried out under essentially physiological conditions and will review briefly some of the possible functions for the protein that have been described.

Setting: Cecilia Makiwane Hospital, Mdantsane, Eastern Cape, Republic of South Africa. Objective: To assess the role of the semi-automated Roche COBAS AMPLICORTMMycobacterium tuberculosis PCR test in the diagnosis of tuberculous meningitis (TBM). Design: Eighty-three specimens of cerebrospinal fluid (CSF) were collected prospectively from 69 patients with suspected TBM. The COBAS AMPLICOR TB PCR test was compared with the manual AMPLICORTMTB PCR test, clinical and cerebrospinal fluid (CSF) findings, direct ZN smear and radiometric TB culture. Results: CSF from 7/40 (17.5%) patients treated for TBM were positive by TB COBAS AMPLICORTM. The sensitivity of the test was not significantly different (p=0.375) from the manual TB AMPLICORTMPCR test. The comparative sensitivities of the TB COBAS AMPLICORTMPCR and the manual AMPLICOR PCR for detecting cases of definite and probable TBM from CSF collected within 9 days of commencing antituberculosis treatment were 40% and 60% respectively. All 29 patients not treated for TBM were negative by COBAS AMPLICORTM, giving a specificity of 100%. Conclusion: The COBAS AMPLICORTMTB PCR test is a rapid and highly specific diagnostic test for TBM. However, there was a non-significant trend favouring slightly greater sensitivity using the manual AMPLICORTMTB PCR test.

Von Ebner gland protein (VEGP) and odorant-binding protein (OBP) were purified from porcine lingual epithelium and nasal mucosa, respectively. Both VEGP and OBP preparations were homogeneous as indicated by SDS–PAGE, isoelectric focusing, gel-filtration and electrospray mass spectrometry. However, high-sensitivity differential scanning calorimetry (HS-DSC) yielded multiphasic denaturation thermograms for both proteins indicating their conformational heterogeneity. The unfolding transition of VEGP is observed at extremely high temperatures (about 110°C), which is unexpected for a protein with significant structural homology to OBP and other lipocalins. Isothermal titration calorimetry (ITC) did not detect the binding of either aspartame or denatonium saccharide to VEGP nor did it detect binding of 2-isobutyl-3-methoxypyrazine (IBMP) to OBP. Extraction of OBP with mixed organic solvents eliminated the conformational heterogeneity and the protein showed a reversible two-state transition in HS-DSC thereafter. ITC also showed that the extracted OBP was able to bind IBMP. These results imply that tightly bound endogenous ligands increase the thermal stability of OBP and block the binding of other ligands. In contrast to OBP, the extraction of VEGP with organic solvents failed to promote binding or to establish thermal homogeneity, most likely because of the irreversible denaturation of VEGP. Thus, the elucidation of the functional behaviour of VEGP is closely related to the exhaustive purging of its endogenous ligands which otherwise very efficiently mask ligand binding sites of this protein.

We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

Bovine β-lactoglobulin has been the subject of intense study over the past 60 years by, effectively, every available physicochemical technique, of which one of the most powerful is X-ray crystallography. We present a short review of the X-ray crystallographic work on milk protein together with an overview of the properties as they are seen from the current state of crystallographic analyses. At the present time, structural analyses do not provide any further insights into the possible function of the protein.

We have produced β-lactoglobulin gene constructs in the pGAPZαA vector for over expression of recombinant wild-type and a putative monomeric mutant protein in Pichia pastoris. Levels of expression of at least 150 mg L ' of the wild-type were achieved in shake flask culture. Ion exchange chromatography showed that the expressed recombinant wild-type β-Lg eluted as a heterogeneous mixture of species, some of which may be owing to small variations in the N-terminus and some to ligand binding. The putative monomeric β-lactoglobulin construct gave an expressed protein that was recognized by anti bovine β-lactoglobulin antibody, but we do not know yet if it forms monomers rather than dimers.

Modelling of the H-bonding interactions involved in the binding of the adenine base component of ATP to E. coli dethiobiotin synthetase (DTBS) suggested that 6-hydroxypyrimidin-4(3H)-one (6-HP4) should selectively bind to this site in the enzyme. Kinetic studies show that 6-HP4 is a competitive inhibitor of DTBS and x-ray crystallography shows that 6-HP4 binds specifically in the purine base binding site of the enzyme.

8-Amino-7-oxononanoate synthase (or 8-amino-7-ketopelargonate synthase; EC 2.3.1.47; AONS) catalyses the decarboxylative condensation of l -alanine and pimeloyl-CoA in the first committed step of biotin biosynthesis. We have cloned, over-expressed and purified AONS from Escherichia coli and determined the crystal structures of the apo and PLP-bound forms of the enzyme. The protein is a symmetrical homodimer with a tertiary structure and active site organisation similar to, but distinct from, those of other PLP-dependent enzymes whose three-dimensional structures are known. The critical PLP-binding lysine of AONS is located at the end of a deep cleft that allows access of the pantothenate arm of pimeloyl-CoA. A cluster of positively charged residues at the entrance to this cleft forms a putative diphosphate binding site for CoA. The structure of E. coli AONS enables identification of the key residues of the PLP-binding site and thus provides a framework with which to understand the biochemical mechanism, which is similar to that catalysed by 5-aminolevulinate synthase and two other α-oxoamine synthases. Although AONS has a low overall sequence similarity with the catalytic domains of other α-oxoamine synthases, the structure reveals the regions of significant identity to be functionally important. This suggests that the organisation of the conserved catalytic residues in the active site is similar for all enzymes of this sub-class of PLP-dependent enzymes and they share a common mechanism. Knowledge of the three-dimensional structure of AONS will enable characterisation of the structural features of this enzyme sub-family that are responsible for this important type of reaction.

beta-Lactoglobulin (beta-Lg) is the major whey protein in ruminant milk and has been implicated in the irreversible denaturation of milk proteins and its associated poor processing behavior during heat treatment. In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure and its behavior in solution, we have prepared and purified 15N-labeled and 13C/15N-double-labelled beta-Lg in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The overexpression of the labeled protein using the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which otherwise interfere with the NMR experiments. At pH 2, the resulting material gives triple resonance NMR spectra of good quality that are consistent with a monomeric, globular protein rich in beta-sheet.

Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate. pH/log Vmax profiles of the enzyme indicate the presence of a single ionizing group with a pKa of 6.2. Chemical modification experiments with diethyl pyrocarbonate have identified the conserved residue His-143 as essential for catalysis in this enzyme and the pKa for this modification is also 6.2, implying that this is the single ionizing residue in dehydroquinase that may be acting as a general base in the catalytic mechanism. Subsequent mutagenesis of this residue (Leech, A. P., James, R., Coggins, J. R., and Kleanthous, C. (1995) J. Biol. Chem. 270, 25827–25836) further suggested that His-143 may be involved in Schiff base formation/breakdown as well as being the proton abstracting general base. The importance of this residue was confirmed by recent x-ray crystallographic data showing His-143 to be at the center of a hydrogen-bonded triad, flanked by the essential Schiff base forming residue Lys-170 and Glu-86. In the present study, we have used mutagenesis and 1H and 13C NMR to assign the resonance of His-143 and probe its ionization state to define more precisely its role in the mechanism of type I dehydroquinase. Following isotopic enrichment of wild-type and H143A dehydroquinase enzymes with [2-13C]histidine, the resonance for His-143 was assigned by comparing their 1H,13C heteronuclear single quantum correlation NMR spectra. pH titrations revealed that whether in the liganded or unliganded state, His-143 does not ionize over the pH range 6–9.5 and so cannot possess a pKa of 6.2. The NMR data are consistent with this residue remaining unprotonated at pH values optimal for the activity of this enzyme (pH > 7). The role of His-143 is re-evaluated in light of these and the recent structural data, and an alternative candidate for the pKa of 6.2 is discussed.

Bovine β-lactoglobulin (β-Lg) is a much studied and commercially important whey protein with an as yet undetermined function, although it is of obvious nutritional value. β-Lg binds a variety of ligands and by comparison of the general structures of these molecules together with several competition studies, it appears that there are at least 3 independent binding sites. In the absence of direct crystallographic evidence, a preliminary modelling study reveals that there is an internal cavity which can readily accommodate retinol in a manner similar to the related lipocalin, retinol-binding protein. On the outer surface, a solvent-accessible hydrophobic cleft runs between the 3-turn α-helix that is packed against the outer surface of the β-barrel. This cleft can accommodate fatty acids like palmitate and stearate. There is difference electron density observed in a soaking experiment with p-nitrophenol at a third site, on the outer surface close to the conserved Trp19/Arg124. This site is large enough to accommodate larger aromatic ligands such as ellipticine, although there is yet no independent evidence for this.

Although beta-lactoglobulin (beta-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine beta-LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of beta-LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a 13C, 15N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric beta-LG A. It includes eight antiparallel beta-strands arranged in a barrel, flanked by an alpha-helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth beta-strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of beta-LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.

The crystal structure of glycyl-L-proline (GLY-PRO) hemihydrate, C(7)H(12)N(2)O(3)(.)0.5H(2)O, has two molecules of GLY-PRO in the asymmetric unit; one molecule adopts the cis configuration at the peptide bond and the other adopts the trans configuration.

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.

Background: β -Lactoglobulin ( β -Lg) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least two pH-dependent conformational changes which may be important. Bovine β -Lg crystallizes in several different lattices, and medium-resolution structures of orthorhombic lattice Y and trigonal lattice Z have been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behaviour of the genetic variants. Results: We have redetermined the structure of β -Lg lattice Z at 3.0 A resolution by multiple isomorphous replacement and have partially refined it (R factor=24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) has been solved by molecular replacement. Refinement of lattice X at 1.8 A resolution gave an R factor of 18.1%. The structure we have determined differs from previously published structures in several ways. Conclusions: Incorrect threading of the sequence in the published structures of β -Lg affects four of the nine β strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the β -Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5°, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the β strand with the free thiol, each of which shows genetic variation.

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS–PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: kcat = 343 and 727 s−1, Km = 0.25 and 0.16 mg mL−1, kcat/Km (specificity constant) = 1374 and 4510 mg mL−1 s−1, respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.

Article; International audience; The interaction between beta-lactoglobulin (BLG) and a newly synthesized Cu(II) complex (2,2'-dibipyridin Cu(II) chloride) was investigated by fluorescence spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC) at temperatures of 27 and 37 A degrees C. The measured heat values of the BLG-Cu(II) complex interaction are reported and analyzed in terms of our previous extended solvation theory for calculating the binding and thermodynamic parameters for the interaction. The Cu(II) complex has a strong ability to quench the intrinsic fluorescence of BLG, to change the microenvironment of tryptophan residues, and to alter the tertiary structure of the protein. Far UV-CD results showed that the complex does not induce any significant changes in the secondary structure of BLG. However, binding of the Cu(II) complex to BLG leads to a significant change in the tertiary structure of BLG, increasing its hydrophobicity and inducing a partial unfolding. This agrees well with ITC data suggesting destabilization of the protein. This finding opens up a way to predict protein destabilization caused by ligand binding, using the extended solvation theory previously proposed.

SummaryThe milk whey protein β-lactoglobulin has been isolated from ovine milk, purified and crystallized in a form suitable for X-ray crystallography. The crystals, which diffract to 1·9 Å resolution, belong to the trigonal space group P3121 with unit cell lengths a = b = 99·8 Å, c = 67·7 Å and unique angle γ = 120·0°. Although there have been many crystal forms reported for bovine β-lactoglobulin, none has been reported with these unit cell parameters. A preliminary native data set has been collected and a molecular replacement solution obtained, using the structure of dimeric bovine β-lactoglobulin as the search model. The importance of the ovine structure in relation to that of the bovine is discussed.

Two structures of bovine odorant binding protein show that this lipocalin is a domain-swapped dimer. The structures stimulate enticing ideas about how this protein transports such a large variety of odorant molecules to the olfactory receptors.

Background: The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N -carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods. Results The first step in catalysis by DTBS is the formation of an enzyme–substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg 2+ dependent. The kinetic constants in the presence and absence of Mg 2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg 2+ , substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the α - and γ -phosphates of ATP in Mg 2+ binding and in the subsequent steps of the reaction. Conclusion Mg 2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS–substrate complex, when Mg 2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.