Membrane interactions of S100A12 (Calgranulin C)

S100A12 (Calgranulin C) is a small acidic calcium-binding peripheral membrane protein with two EF-hand structural motifs. It is expressed in macrophages and lymphocytes and highly up-regulated in several human inflammatory diseases. In pigs, S100A12 is abundant in the cytosol of granulocytes, where it is believed to be involved in signal modulation of inflammatory process. In this study, we investigated the interaction of the porcine S100A12 with phospholipid bilayers and the effect that ions (Ca2+, Zn2+ or both together) have in modifying protein-lipid interactions. More specifically, we intended to address issues such as: (1) is the protein-membrane interaction modulated by the presence of ions? (2) is the protein overall structure affected by the presence of the ions and membrane models simultaneously? (3) what are the specific conformational changes taking place when ions and membranes are both present? (4) does the protein have any kind of molecular preferences for a specific lipid component? To provide insight into membrane interactions and answer those questions, synchrotron radiation circular dichroism spectroscopy, fluorescence spectroscopy, and surface plasmon resonance were used. The use of these combined techniques demonstrated that this protein was capable of interacting both with lipids and with ions in solution, and enabled examination of changes that occur at different levels of structure organization. The presence of both Ca2+ and Zn2+ ions modify the binding, conformation and thermal stability of the protein in the presence of lipids. Hence, these studies examining molecular interactions of porcine S100A12 in solution complement the previously determined crystal structure information on this family of proteins, enhancing our understanding of its dynamics of interaction with membranes

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of E.coli

Background: Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria. Methods: The ß-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the E.coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined. Results: Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold. Conclusions: The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. General Significance: We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors

Temporal dynamics of the shrub and herbaceous layer of an area of moist grassland in Alto Paraíso de Goiás, Brazil

Este trabalho avaliou a dinâmica estrutural e fl orística de uma comunidade de espécies herbáceo-arbustivas de uma área de campo limpo úmido em Alto Paraíso de Goiás, o primeiro inventário realizado em 2000 (T0) e o segundo em 2007 (T1). A diversidade de Shannon entre os períodos foi comparada pelo teste-t de Hutcheson e a similaridade fl orística, pelo índice de similaridade de Chao-Sørensen. As relações fl orísticas e a cobertura, entre os períodos e as linhas, foram avaliadas por meio de análises de correspondência retifi cada (DCA). Foram amostradas 98 espécies, 88 no T0 e 67 no T1, sendo 31 exclusivas do T0 e 10 do T1. A diversidade fl orística na comunidade foi elevada nos dois períodos, porém diferente entre esses (t = 7,12; p < 0,001), devido a variação no número e cobertura das espécies. A similaridade entre os dois inventários foi alta (Chao-Sørensen ± IC = 0,841 ± 0,074). A ordenação por DCA indicou relações entre a composição fl orística e a cobertura com o gradiente de umidade e de matéria orgânica no solo identifi cados em T0. Houve modifi cações nas linhas em zonas sazonais, as quais se tornaram mais semelhantes às linhas constantemente saturadas por água. Em um intervalo de sete anos o campo limpo úmido apresentou mudanças na composição fl orística e, principalmente na estrutura devido o aumento da cobertura de espécies perenes, cespitosas e entouceiradas, que foram favorecidas pela maior umidade no solo em resposta à elevação da pluviosidade da região. __________________________________________________________________________________________ ABSTRACTTh is study evaluated the fl oristic and structural dynamics of a community of herbaceous-shrub species in an area of moist grassland in Alto Paraíso de Goiás. Th e fi rst inventory was undertaken in 2000 (T0) and the second in 2007 (T1). Shannon’s diversity between the periods was compared by Hutchesons´s t-test, and the fl oristic similarity by the Chao-Sørensen similarity index. Floristic composition and cover, between periods and lines, were evaluated by detrended correspondence analysis (DCA). We sampled 98 species, 88 at T0 and 67 at T1; 31 were unique to T0 and 10 to T1. Floristic diversity in the community was high in both periods, but diff erent between them (t = 7.12, p <0.001), due to variation in species number and coverage. Similarity between the two surveys was high (Chao-Sørensen CI = ± 0.841 ± 0.074). Th e DCA ordination indicated relationships between the fl oristic composition and cover with a gradient of moisture and organic matter in the soil identifi ed in T0. Th ere were changes in the lines in the seasonal zones, which became more similar in those constantly saturated with water. During an interval of seven years the moist grassland showed changes in fl oristic composition and mainly in structure due to increased cover of the clumped tussock perennial species, which were favored by higher soil moisture due to high rainfall in the region

Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: applications in secondary structure analyses

Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically-defined spectroscopic signatures derived from proteins with known three-dimensional structures. Many proteins identified as being “Intrinsically Disordered Proteins” have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as “other”, “random coil”, “unordered” or “disordered”. However the “other” category can also include polyprolineII (PPII)-type structures, whose spectral properties have not been well-distinguished from those of unordered structures. In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII-type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid-containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220 nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins