Hydrogen exchange in a large 29 kD protein and characterization of molten globule aggregation by NMR

The nature of denatured ensembles of the enzyme human carbonic anhydrase (HCA) has been extensively studied by various methods in the past. The protein constitutes an interesting model for folding studies that does not unfold by a simple two-state transition, instead a molten globule intermediate is highly populated at 1.5 M GuHCl. In this work, NMR and H/D exchange studies have been conducted on one of the isozymes, HCA I. The H/D exchange studies, which were enabled by the previously obtained resonance assignment of HCA I, have been used to identify unfolded forms that are accessible from the native state. In addition, the GuHCl-induced unfolded states of HCA I have also been characterized by NMR at GuHCl concentrations in the 0-5 M range. The most important findings in this work are as follows: (1) Amide protons located in the center of the beta-sheet require global unfolding events for efficient H/D exchange. (2) The molten globule and the native state give similar protection against H/D exchange for all of the observable amide protons (i.e., water seems not to efficiently penetrate the interior of the molten globule). (3) At high protein concentrations, the molten globule can form large aggregates, which are not detectable by solution-state NMR methods. (4) The unfolded state (U), present at GuHCl concentrations above 2 M, is composed of an ensemble of conformations having residual structures with different stabilities.     

Reactions of Mycobacterium tuberculosis truncated hemoglobin O with ligands reveal a novel ligand-inclusive hydrogen bond network

Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O(2) and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 microM(-1) s(-1)) and dissociation (<0.006 s(-1)) of O(2) are among the slowest known, precluding transport or diffusion of O(2) as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25- and 77-fold increases in the combination rate constants for O(2) and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the chi((2,1)) dihedral angle for the indole ring increases progressively from approximately 93 degrees to at least 100 degrees in going sequentially from the deoxy to CO to O(2) derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O(2) dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.     

SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of approximately 80 fF that plateaued within approximately 200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K(d) values were 6 and 29 micro M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1(-/-) mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1(-/-) mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl(-) into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane K(ATP)-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca(2+)-induced exocytosis.     

Comparative non-shivering thermogenesis in adjacent populations of the common spiny mouse (Acomys cahirinus) from opposite slopes: the effects of increasing salinity

We compared non-shivering thermogenesis between two adjacent populations of the common spiny mouse Acomys cahirinus from different habitats, in relation to increasing salinity. Individuals were captured from the north- and south-facing slopes of the same valley, that represent "Mediterranean" and "desert" habitats, respectively. We hypothesized that the two populations of mice would differ in their thermoregulatory capacities, reflecting their need to cope with the environmental stress in each habitat. We measured resting metabolic rate by recording oxygen consumption, body temperature and response to an injection of exogenous noradrenaline. Mice were maintained on diets with increasing levels of salt intake to examine their abilities to cope with increasing osmotic stress. Mice from north-facing slopes generally had a higher resting metabolic rate and a higher increase in oxygen consumption in response to noradrenaline than mice from south-facing slopes. Increasing salinity decreased resting metabolic rate values, body temperature, and oxygen consumption in response to noradrenaline in both populations, and diminished slope-dependant differences. We suggest that these differences could be a result of an ongoing adaptive process to different climatic conditions, typical of the Mediterranean region, that are a demonstrable example of evolution in action.     

Large-scale mirror-symmetry organization of human occipito-temporal object areas

We have combined functional maps of retinotopy (eccentricity and meridian mapping), object category, and motion in a group of subjects to explore the large-scale topography of higher-order object areas. Our results reveal seven consistent category-related entities situated in the occipito-temporal cortex adjoining early visual areas. These include two face-related regions, three object-related regions, and two building-related regions. Interestingly, this complex category-related pattern is organized in a large-scale dorso-ventral mirror symmetry of object category. Furthermore, correlating this pattern to the map of visual field eccentricity, we found that the entire network of areas could be related to a single and unified eccentricity map. We hypothesize that this large-scale organization points to a possible development of high-order object areas through extension and specialization of a single proto-representation.     

Transient interaction with nanoparticles "freezes" a protein in an ensemble of metastable near-native conformations

It is well-known that adsorption of proteins on interfaces often induces substantial alterations of the protein structure. However, very little is known about whether these conformational changes have any consequence for the protein conformation after desorption from the interface. To investigate this matter, we have selected a protein-particle system in which the enzyme human carbonic anhydrase I (HCAI) alternates between the adsorbed and free state upon interaction with the silica nanoparticles. High-resolution NMR analysis of the protein with the particles present in the sample shows a spectrum that indicates a molten globular-like structure. Removal of particles results in refolding of virtually all HCAI molecules to a fully active form. However, the two-dimensional NMR analysis shows that refolding does not result in a single well-defined protein structure but rather provides an ensemble of protein molecules with near-native conformations. A detailed comparative chemical shift analysis of 108 amide signals in (1)H-(15)N HSQC spectra of native and desorbed HCAI reveals that the most profound effects are located at beta-strands in the center of the molecule. The observation of very slow H-D exchange in the central beta-strands of HCAI [Kjellsson, A., Sethson, I., and Jonsson, B. H. (2003) Biochemistry 42, 363-374] in conjunction with our results indicates that the kinetic barriers for conformational rearrangements in the central core of the protein are low in the presence of nanoparticles but are very high under native conditions.     

Fluorometric TLC assay for evaluation of protein kinase inhibitors

A fluorometric assay for measuring protein kinase activity has been developed. The assay is based on the separation of fluorescently marked substrate 5-carboxytetramethylrhodamine-kemptide (5-TAMRA-kemptide) from its phosphorylated counterpart by TLC and quantification of the product ratiometrically by fluorescence imaging. The utility of the assay was demonstrated by measuring the activity of cAMP-dependent protein kinase. 5-TAMRA-kemptide was characterized as a substrate of this kinase by the kinetic parameters K(m)(app) and V(max). The attachment of 5-TAMRA dye to the N terminal of kemptide decreased the K(m)(app) value but did not have a significant effect on the rate and stoichiometry of the phosphorylation reaction. The inhibitory potency of three known inhibitors was evaluated with the new assay. The closeness of the obtained inhibitory activities of the compounds to the activities determined with the phosphocellulose paper-binding assay, as well as the Z' factor value of 0.5, demonstrates the reliability of the new assay for evaluation of inhibitors of protein kinases.     

Vertical Distribution of Zooplankton in a Strongly Stratified Hypertrophic Lake

The vertical and temporal distribution of metazooplankton in the small hypertrophic, strongly stratified, temperate Lake Verevi (Estonia) was studied during 1998–2001. The zooplankton of Lake Verevi is characteristic of hypertrophic lakes, with a small number of dominant species, rotifers being the main ones, and juveniles prevailing among copepods. In 1999–2001, the average abundance of metazooplankton in the lake was 1570 × 10³ ind m⁻³; in the epilimnion 2320 × 10³ ind m⁻³, in the metalimnion 2178 × 10³ ind m⁻³, and in the hypolimnion 237 × 10³ ind m⁻³. The average biomass of metazooplankton was 1.75 g m⁻³; in the epi-, meta- and hypolimnion, accordingly, 2.16, 2.85 and 0.26 g m⁻³. The highest abundances – 19,136 × 10³ ind m⁻³ and 12,008 × 10³ ind m⁻³ – were registered in the lower half of the metalimnion in 24 May and 5 June 2001, respectively. Rotifer Keratella cochlearis f. typica (Gosse, 1851) was the dominating species in abundance. In biomass, Asplanchna priodonta Gosse, 1850, among the rotifers, and Eudiaptomus graciloides (Lilljeborg, 1888), among the copepods, dominated. According to the data from 2000–2001, the abundance and biomass of both copepods and rotifers were highest in spring. Zooplankton was scarce in the hypolimnion, and no peaks were observed there. During the summers of 1998 and 1999, when thermal stratification was particularly strong, zooplankton was the most abundant in the upper half of the metalimnion, and a distinct peak of biomass occurred in the second fourth of the metalimnion. Probably, the main factors affecting the vertical distribution of zooplankton in L. Verevi are fish, Chaoborus larvae, and chemocline, while food, like phytoplankton, composition and abundance may affect more the seasonal development of zooplankton.     

Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels

Oxygen binding curves of sol-gel-encapsulated deoxy human adult hemoglobin (HbA) have previously revealed two distinct noncooperative populations with oxygen binding affinities approximately 1000 and 100 times lower than that of the high-affinity R state. The two populations which have been termed the low-affinity (LA) and high-affinity (HA) T states can be selectively stabilized using two different encapsulation protocols for deoxy-HbA. The present study seeks to understand the factors giving rise to these different affinity states. Visible and UV resonance Raman spectroscopies are used to characterize the conformational properties of both the deoxy and deoxy-turned-carbonmonoxy (CO) derivatives of HbA derived from the two encapsulation protocols. The geminate and bimolecular recombination of CO to the photodissociated CO derivatives is used to characterize the functional properties of the slowly evolving encapsulated populations. The results show that the initial deoxy-HbA populations are conformationally indistinguishable with respect to encapsulation protocol. The addition of CO to sol-gel-encapsulated deoxy-HbA triggers a detectable progression of conformational and functional changes. Visible resonance Raman spectra of the CO photoproduct reveal a progression of changes of the iron-proximal histidine stretching frequencies: 215, 222, 227, and 230 cm(-1). The low and high values correspond to the initial deoxy T state and liganded R (R(2)) state species, respectively. The 222 and 227 cm(-1) species are generated using encapsulation protocols that give rise to what are termed the LA and HA T states, respectively. The UV resonance Raman spectra of these and related species indicate that the progression from deoxy T to LA to HA is associated with a progressive loosening of T state constraints within the hinge and switch regions of the alpha(1)beta(2) interface. The time scale for the progression is determined by a balance between the ligation-initiated evolution toward high-affinity conformations and factors such as allosteric effectors, gel matrix, and added glycerol that slow ligand-binding-induced relaxation. Thus, it appears that the encapsulation protocol-dependent rate of ligand-binding-induced relaxation determines the functional properties of the initially encapsulated deoxy-HbA population.     

Effects of ghrelin on insulin and glucagon secretion: a study of isolated pancreatic islets and intact mice

We combined in vitro and in vivo methods to investigate the effects of ghrelin, a novel gastric hormone, on insulin and glucagon release. Studies of isolated mouse islets showed that ghrelin concentrations in the physiological range (0.5-3 nmol l(-1)) had no effect on glucose-stimulated insulin release, while low ghrelin concentrations (1-100 pmol l(-1)) inhibited and high (0.1 and 1 micromol l(-1)) stimulated. The insulin response to glucose was enhanced in the presence of a high ghrelin concentration (100 nmol l(-1)). Glucagon release was stimulated by ghrelin (0.1 pmol l(-1) to 1 micromol l(-1)); this effect was maintained in the presence of glucose (0-20 mmol l(-1)). In intact mice, basal plasma insulin was suppressed by 1 and 10 nmol kg(-1) of ghrelin, 2 and 6 min after i.v. injection. Ghrelin (0.2-10 nmol kg(-1) i.v.) suppressed also the glucose-stimulated insulin response and impaired the glucose tolerance (at a ghrelin dose of 3.3 nmol kg(-1)). Ghrelin (1 or 10 nmol kg(-1) i.v.) inhibited the insulin response to the phospholipase C stimulating agent carbachol and enhanced the insulin response to the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX) but did not affect the response to the membrane-depolarizing amino acid l-arginine. These observations suggest that the inhibitory effect of ghrelin on glucose-induced insulin release is in part exerted on phospholipase C pathways (and not on Ca(2+)entry), while the stimulatory effect of high doses of ghrelin depends on cyclic AMP. In contrast to the spectacular glucagon-releasing effect of ghrelin in vitro, ghrelin did not raise plasma glucagon. Carbachol, IBMX and l-arginine stimulated glucagon release. These responses were impaired by ghrelin, suggesting that it suppresses the various intracellular pathways (phospholipase C, cyclic AMP and Ca(2+)), that are activated by the glucagon secretagogues. Together these observations highlight (but do not explain) the different effects of ghrelin on glucagon release in vitro and in vivo. The results show that ghrelin has powerful effects on islet cells, suggesting that endogenous ghrelin may contribute to the physiological control of insulin and glucagon release. However, the narrow "window" of circulating ghrelin concentrations makes this doubtful.