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1.
The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1-84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1-84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce alpha-helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed.  相似文献   

2.
Liang ZX  Tsigos I  Lee T  Bouriotis V  Resing KA  Ahn NG  Klinman JP 《Biochemistry》2004,43(46):14676-14683
The psychrophilic alcohol dehydrogenase (psADH) cloned from Antarctic Moraxella sp. TAE123 exhibits distinctive catalytic parameters in relation to the homologous thermophilic alcohol dehydrogenase (htADH) from Bacillus stearothermophilus LLD-R. Amide hydrogen-deuterium (H/D) exchange studies using Fourier-transformed infrared (FTIR) spectroscopy and mass spectrometry (MS) were conducted to investigate whether the differences are caused by variation in either global or regional protein flexibility. The FTIR H/D exchange study suggested that psADH does not share similar global flexibility with htADH at their physiologically relevant temperatures as has been predicted by the "corresponding state" hypothesis. However, the MS H/D exchange study revealed a more complicated picture concerning the flexibility of the two homologous enzymes. Analysis of the deuteration and exchange rates of protein-derived peptides suggested that only some functionally important regions in psADH that are involved in substrate and cofactor binding exhibit greater flexibility compared to htADH at low temperature (10 degrees C). These observations strongly suggest that variable conformational flexibility between the two protein forms is a local phenomenon, and that global H/D exchange measurement by FTIR can be misleading and should be used with discretion. These results are supportive of the idea that functionally important local flexibility can be uncoupled from global thermal stability. The structural factors underlying the differences in local protein flexibility and catalysis between htADH and psADH are discussed in conjunction with results from crystallographic and fluorescence spectroscopy studies.  相似文献   

3.
4.
An understanding of the structure-function relationship of proteins under different chemical-physical conditions is of fundamental importance for an understanding of their structure and function in cells. In this paper we report the effects of sodium dodecyl sulfate and temperature on the structure of beta-galactosidase from Escherichia coli, as monitored by fluorescence spectroscopy. The structure of the protein was studied in the temperature range of 10-60 degrees C in the absence and presence of sodium dodecyl sulfate by frequency-domain measurement of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that at 10 degrees C the tryptophanyl emission decays were well described by a three exponential decays model, and that the temperature increase resulted in shortening of the long-lived component with little change in the short- and middle-lived components. The addition of SDS to the protein solution also affected the long-lived component. The effects of the detergent and temperature on the enzyme structure were also investigated by means of quenching experiments and anisotropy decays. The obtained results showed that the presence of SDS confers more flexibility to the protein structure, and suggest a strict relation between enzyme activity and protein flexibility.  相似文献   

5.
The ‘Moran effect’ predicts that dynamics of populations of a species are synchronized over similar distances as their environmental drivers. Strong population synchrony reduces species viability, but spatial heterogeneity in density dependence, the environment, or its ecological responses may decouple dynamics in space, preventing extinctions. How such heterogeneity buffers impacts of global change on large‐scale population dynamics is not well studied. Here, we show that spatially autocorrelated fluctuations in annual winter weather synchronize wild reindeer dynamics across high‐Arctic Svalbard, while, paradoxically, spatial variation in winter climate trends contribute to diverging local population trajectories. Warmer summers have improved the carrying capacity and apparently led to increased total reindeer abundance. However, fluctuations in population size seem mainly driven by negative effects of stochastic winter rain‐on‐snow (ROS) events causing icing, with strongest effects at high densities. Count data for 10 reindeer populations 8–324 km apart suggested that density‐dependent ROS effects contributed to synchrony in population dynamics, mainly through spatially autocorrelated mortality. By comparing one coastal and one ‘continental’ reindeer population over four decades, we show that locally contrasting abundance trends can arise from spatial differences in climate change and responses to weather. The coastal population experienced a larger increase in ROS, and a stronger density‐dependent ROS effect on population growth rates, than the continental population. In contrast, the latter experienced stronger summer warming and showed the strongest positive response to summer temperatures. Accordingly, contrasting net effects of a recent climate regime shift—with increased ROS and harsher winters, yet higher summer temperatures and improved carrying capacity—led to negative and positive abundance trends in the coastal and continental population respectively. Thus, synchronized population fluctuations by climatic drivers can be buffered by spatial heterogeneity in the same drivers, as well as in the ecological responses, averaging out climate change effects at larger spatial scales.  相似文献   

6.
7.
Acidic fibroblast growth factor-1 (FGF-1) is an angiogenic protein which requires binding to a polyanion such as heparin for its mitogenic activity and physicochemical stability. To evaluate the extent to which this heparin dependence on solution stability could be reduced or eliminated, the structural integrity and conformational stability of 10 selected FGF-1 mutants were examined as a function of solution pH and temperature by a series of spectroscopic methods including circular dichroism, intrinsic and extrinsic fluorescence spectroscopy and static light scattering. The biophysical data were summarized in the form of colored empirical phase diagrams (EPDs). FGF-1 mutants were identified with stability profiles in the absence of heparin comparable to that of wild-type FGF-1 in the presence of heparin while still retaining their biological activity. In addition, a revised version of the EPD methodology was found to provide an information rich, high throughput approach to compare the effects of mutations on the overall conformational stability of proteins in terms of their response to environmental stresses such as pH and temperature.  相似文献   

8.
9.
Prasad S  Mitra S 《Biochemistry》2002,41(49):14499-14508
The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site. We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes. The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.  相似文献   

10.
We applied x-ray diffraction, calorimetry, and infrared spectroscopy to lipid mixtures of palmitoyl-oleoyl phosphatidylcholine, sphingomyelin, and ceramide. This combination of experimental techniques allowed us to probe the stability and structural properties of coexisting lipid domains without resorting to any molecular probes. In particular, we found unstable microscopic domains (compositional/phase fluctuations) in the absence of ceramide, and macroscopically separated fluid and gel phases upon addition of ceramide. We also observed phase fluctuations in the presence of ceramide within the broad phase transition regions. We compare our results with fluorescence spectroscopy data and complement the previously reported phase diagram. We also obtained electron paramagnetic resonance data to assess the possible limitations of techniques employing a single label. Our study demonstrates the necessity of applying a combination of experimental techniques to probe local/global structural and fast/slow motional properties in complex lipid mixtures.  相似文献   

11.
Bovine glutamate dehydrogenase (GDH) is allosterically regulated and requires substrate‐induced subunit interactions for maximum catalytic activity. Steady‐state and presteady‐state kinetics indicate that the rate‐limiting step depends on the nature of the substrate and are likely associated with conformational fluctuations necessary for optimal hydride transfer. Deuterated glutamate shows a steady‐state isotope effect but no effect on the presteady‐state burst rate, demonstrating that conformational effects are rate limiting for hydride transfer while product release is overall rate limiting for glutamate. Guanidine hydrochloride unfolding, heat inactivation, and differential scanning calorimetry demonstrate the effects of alternative substrates, glutamate and norvaline, on conformational stability. Glutamate has little effect on overall stability, whereas norvaline markedly stabilizes the protein. Limited proteolysis demonstrates that glutamate had a variety of effects on local flexibility, whereas norvaline significantly decreased conformational fluctuations that allow protease cleavage. Dynamic light scattering suggests that norvaline stabilizes all interfaces in the hexamer, whereas glutamate had little effect on trimer–trimer interactions. The substrate glutamate exhibits negative cooperativity and complex allosteric regulation but has only minor effects on global GDH stability, while promoting certain local conformational fluctuations. In contrast, the substrate norvaline does not show negative cooperativity or allow allosteric regulation. Instead, norvaline significantly stabilizes the enzyme and markedly slows or prevents local conformational fluctuations that are likely to be important for cooperative effects and to determine the overall rate of hydride transfer. This suggests that homotropic allosteric regulation by the enzymatic substrate involves changes in both global stability and local flexibility of the protein.  相似文献   

12.
In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled.  相似文献   

13.
It is widely accepted that species diversity is contingent upon the spatial scale used to analyze patterns and processes. Recent studies using coarse sampling grains over large extents have contributed much to our understanding of factors driving global diversity patterns. This advance is largely unmatched on the level of local to landscape scales despite being critical for our understanding of functional relationships across spatial scales. In our study on West African bat assemblages we employed a spatially explicit and nested design covering local to regional scales. Specifically, we analyzed diversity patterns in two contrasting, largely undisturbed landscapes, comprising a rainforest area and a forest‐savanna mosaic in Ivory Coast, West Africa. We employed additive partitioning, rarefaction, and species richness estimation to show that bat diversity increased significantly with habitat heterogeneity on the landscape scale through the effects of beta diversity. Within the extent of our study areas, habitat type rather than geographic distance explained assemblage composition across spatial scales. Null models showed structure of functional groups to be partly filtered on local scales through the effects of vegetation density while on the landscape scale both assemblages represented random draws from regional species pools. We present a mixture model that combines the effects of habitat heterogeneity and complexity on species richness along a biome transect, predicting a unimodal rather than a monotonic relationship with environmental variables related to water. The bat assemblages of our study by far exceed previous figures of species richness in Africa, and refute the notion of low species richness of Afrotropical bat assemblages, which appears to be based largely on sampling biases. Biome transitions should receive increased attention in conservation strategies aiming at the maintenance of ecological and evolutionary processes.  相似文献   

14.
Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins.  相似文献   

15.
The effects of several polyanions on the hydrolysis of the chromogenic substrate L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroaniline (S-2366) and on the activation of factor IX by factor XIa have been investigated. Two forms of dextran sulfate (M(r) approximately 500000 and M(r) approximately 10000, DX10) and two forms of heparin (64 disaccharide units, M(r) approximately 14000, and hypersulfated heparin, S-Hep, M(r) approximately 12000) inhibited both factor XIa amidolytic activity and factor IX activation in a concentration-dependent manner. The inhibitory effect was not due to binding of either substrate by the polyanions since only a decrease in V(max) without any effect on K(m) was observed in kinetic assays. Steric inhibition is unlikely since the concentrations of polyanions required for inhibition of small peptide hydrolysis were lower than those required for macromolecular substrate cleavage. In contrast, an allosteric inhibitory mechanism was supported by an enhancement of the dansyl fluorescence of 5-(dimethylamino)-1-(naphthalenesulfonyl)glutamylglycylarginyl- (DEGR-) factor XIa observed when the fluorophore was in complex with either DX10 or S-Hep. Moreover, in the presence of a polyanion the fluorophore was far more resistant to quenching by acrylamide. These results provide compelling evidence that factor XIa binding to the polyanions, dextran sulfate and heparin, results in inhibition of the enzyme by an allosteric mechanism.  相似文献   

16.
Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).  相似文献   

17.
Sucrose is a natural osmolyte accumulated in cells of organisms as they adapt to environmental stresses. In vitro, sucrose increases protein stability and forces partially unfolded structures to refold. Its effects on the native fold structure and dynamics are not fully established. This study, utilizing Trp phosphorescence spectroscopy, examined the influence of molar concentrations of sucrose on the flexibility of metal-free azurin from Pseudomonas aeruginosa. In addition, by means of specific mutants of the test protein, namely I7S, F110S, and C3A/C26A, that altered its thermodynamic stability, its intrinsic flexibility, and the extent of internal hydration, this investigation sought to identify possible correlations between these features of protein structure and the influence of the osmolyte on protein dynamics. Alterations of structural fluctuations were assessed by both the intrinsic phosphorescence lifetime (tau), which reports on local structure about the triplet probe, and the acrylamide bimolecular quenching rate constant (k(q)) that is a measure of the average acrylamide diffusion coefficient through the macromolecule. From the modulation of tau and k(q) across a wide temperature range and up to a concentration of 2M sucrose, it is concluded that sucrose attenuates structural fluctuations principally when macromolecules are internally hydrated and thermally expanded. Preliminary tests with trehalose and xylitol suggest that the effects of sucrose are general of the polyol class of osmolytes.  相似文献   

18.
In this study the relative importance of valence and charge density of the polycation chitosan on the compaction process of DNA and xanthan is investigated. Chitosans with approximately equal valence but differing in their charge density were employed to form polyelectrolyte complexes with the two polyanions. The resulting structures (toroids, rods, and globules) have been visualized by AFM. For DNA-chitosan the complexation process was additionally studied by utilizing the fluorescent probe ethidium bromide. The results show that not only the total charge per chitosan molecule (valence), but also the charge density is important in determining the association with polyanions such as DNA and xanthan. Furthermore, it is demonstrated that the pH at which the complexation takes place is an important parameter in the complexation process, influencing the structures formed.  相似文献   

19.
The Moran effect for populations separated in space states that the autocorrelations in the population fluctuations equal the autocorrelation in environmental noise, assuming the same linear density regulation in all populations. Here we generalize the Moran effect to include also nonlinear density regulation with spatial heterogeneity in local population dynamics as well as in the effects of environmental covariates by deriving a simple expression for the correlation between the sizes of two populations, using diffusion approximation to the theta-logistic model. In general, spatial variation in parameters describing the dynamics reduces population synchrony. We also show that the contribution of a covariate to spatial synchrony depends strongly on spatial heterogeneity in the covariate or in its effect on local dynamics. These analyses show exactly how spatial environmental covariation can synchronize fluctuations of spatially segregated populations with no interchange of individuals even if the dynamics are nonlinear.  相似文献   

20.
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, combined with hydrogen/deuterium exchange technique and time-resolved fluorescence spectroscopy, has been used to investigate the changes in structure and dynamics that underlie the thermodynamic stability differences observed for three closely homologous proteins: dendrotoxins I and K, and bovine pancreatic trypsin inhibitor (BPTI). The experiments were performed on proteins under their native state and a modified form, obtained by selective reduction of a disulfide bond at the surface of the molecule, increasing slightly the backbone flexibility without changing the average structure. The data confirmed the high local as well as global rigidity of BPTI. In protein K, the exchange process was slow during the first 2 h of exchange, presumably reflecting a compact three-dimensional conformation, and then increased rapidly, the internal amide protons of the beta-strands exchanging 10-fold faster than in BPTI or protein I. The most probable destabilizing element was identified as Pro32, in the core of the beta-sheet. Protein I was found to present a 10% more expanded volume than protein K or BPTI, and there is a possible correlation between the resulting increased flexibility of the molecule and the lower thermodynamic stability observed for this protein. Interestingly, the interior amide protons of the beta-sheet structure were found to be as protected against exchange in protein I as in BPTI, suggesting that, although globally more flexible than that of Toxin K or BPTI, the structure of Toxin I could be locally quite rigid. The structural factors suspected to be responsible for the differences in internal flexibility of the two toxins could play a significant role in determining their functional properties.  相似文献   

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