An automatic tool to facilitate the statistical group analysis of DTI.

BACKGROUND: Users may have difficulty calculating DTI group statistics since they need to master several complex tools that require high user intervention. A tool called DTIStatistics for the automatic and easy calculation of DTI group statistics was developed to reduce analysis times and possible errors. METHODS: The proposed software was designed by using a user-centred methodology in which we performed an iterative usability evaluation with an expert committee. Once the experts׳ requirements were fulfilled, we performed a validation of the final version of DTIStatistics with target users, comparing the execution time of this tool and the standard pipeline normally used. RESULTS: Target users needed significantly less time to complete the tasks with DTIStatistics, reducing the analysis time from 1383.78 to 57.2s. They were able to complete all the tasks and barely made errors. Moreover, target users were not able to display the analysis results with the standard pipeline, but when using our tool they only needed 34s. Target users found DTIStatistics easy to learn, use and interact with, and they concluded that they could effectively complete the tasks with it. Additionally, we present example results in the study of depression to demonstrate the validity of DTIStatistics for clinical research. CONCLUSIONS: DTIStatistics facilitates and significantly automates the calculation of DTI group statistics by reducing the analysis times, which implies lower costs. DTIStatistics is highly applicable in clinical research, as demonstrated by the fact that it is currently being used at the University Hospital, University of Navarra (Spain).

Distortion correction and calibration of intra-operative spine X-ray images using a constrained DLT algorithm.

This work presents an automatic method for distortion correction and calibration of intra-operative spine X-ray images, a fundamental step for the use of this modality in computer and robotic assisted surgeries. Our method is based on a prototype calibration drum, attached to the c-arm intensifier during the intervention. The projections of its embedded fiducial beads onto the X-ray images are segmented by the proposed method, which uses its calculated centroids to undo the distortion and, afterwards, calibrate the c-arm. For the latter purpose, we propose the use of a constrained version of the well known Direct Linear Transform (DLT) algorithm, reducing its degrees of freedom from 11 to 3. Experimental evaluation of our method is included in this work, showing that it is fast and more accurate than other existing methods. The low segmentation error level also ensures accurate calibration of the c-arm, with an expected error of 4% in the computation of its focal distance.

Thermal adaptation of heat shock proteins.

Heat shock proteins (Hsps) are molecular chaperones that oppose stress-induced denaturation of other proteins. Hsps are present in all organisms. Apart from assisting in the efficient folding of newly synthesized proteins they maintain pre-existing proteins in a stable conformation, preventing their aggregation, under stress conditions. The latter role, essential for thermal adaptation, requires that the chaperone system change from a folding to a storing function at heat shock temperatures. The temperature at which this change occurs depends on the presence of a thermosensor in at least one of the components of the chaperone systems. In this review, we focus on the bacterial GroE and DnaK systems, describe their temperature-sensitive protein components, and the location of the thermosensor within the structure of these components. While the thermosensor of the GroE system is located at the inter-ring interface of GroEL, that of the DnaK system occurs in its co-chaperone GrpE. Analysis of these examples demonstrates the amazing mechanistic diversity of thermal stress adaptation and of functional convergence of structurally unrelated proteins.

Structural and functional properties of Escherichia coli-derived nucleoplasmin. A comparative study of recombinant and natural proteins.

Fourier transform infrared spectroscopy, circular dichroism and prediction techniques have been used to investigate the conformational properties of nucleoplasmin isolated from oocytes and eggs of Xenopus. laevis and overexpressed in Escherichia coli. A simple and fast method allows purification of recombinant nucleoplasmin free of truncated and/or aggregated forms, and therefore provides a suitable sample to carry out the structural and functional comparison between these proteins. The secondary structure of the three proteins estimated from both spectroscopic techniques was very similar, and was found to be 31--33% loops, 27--34% beta structure, 22--26% turns and 9-14% alpha helix. Prediction studies, in good agreement with experimental data, also suggest that beta structure is the major regular conformation, and that loops and turns are the most abundant conformational features within the secondary structure of nucleoplasmin. Furthermore, the spectroscopic characterization of a truncated version of the protein, lacking 80 residues at the C-terminus, and the prediction data indicate that the secondary structure elements of the protein are segregated into two regions. The N-terminal fragment (comprising residues 1--120) which holds all the putative beta strands, and the solvent-exposed C-terminal region, that is suggested to be enriched in turn and loop structures. The phosphate/protein monomer molar ratios, obtained from chemical analysis and mass spectrometry, are 0, 3 and 7--10 for recombinant, oocyte and egg nucleoplasmin, respectively. Phosphorylation does not significantly affect the secondary structure of the protein, but clearly modulates its ability to decondense sperm nuclei and to remove basic proteins from DNA.

Excluded volume effects on the refolding and assembly of an oligomeric protein. GroEL, a case study.

We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers. The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly. Crowding modulates GroEL oligomerization in two different ways. First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium. Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation. This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits. If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo.

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability.

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.

Exogenously incorporated ketocarotenoids in large unilamellar vesicles. Protective activity against peroxidation.

The ability of astaxanthin and canthaxanthin as chain-breaking antioxidants was studied in Cu(2+)-initiated peroxidation of phosphatidylcholine large unilamellar vesicles (LUVs). Both carotenoids increased the lag period that precedes the maximum rate of lipid peroxidation, though astaxanthin showed stronger activity. For these experiments, different amounts of xanthophylls were exogenously added to previously made LUVs, non-incorporated pigment being afterwards removed. Differential scanning calorimetry assays with L-beta,gamma-dimyristoyl-alpha-phosphatidylcholine LUVs demonstrated that xanthophylls incorporated as described interact with the lipid matrix becoming interspersed among the phospholipid molecules.

Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection.

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-Lerythro-5,6,7, 8-tetrahydrobiopterin (H4biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 +/- 4 microM). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 +/- 2 microM) and the negative cooperativity was amplified (h = 0.27 +/- 0.03). The dimeric C-terminal deletion mutant (Delta473-528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; Km = 62.7 +/- 5.7 microM) and 3-methyl-5,6,7, 8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 +/- 0.1; Km = 687 +/- 50 microM). In the presence of 1 mM H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (DeltaRU) response with negative cooperativity (h = 0.52-0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo-and the holo-hTH1 on binding of H4biopterin and Lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.

Conformational changes generated in GroEL during ATP hydrolysis as seen by time-resolved infrared spectroscopy.

Changes in the vibrational spectrum of the chaperonin GroEL in the presence of ADP and ATP have been followed as a function of time using rapid scan Fourier transform infrared spectroscopy. The interaction of nucleotides with GroEL was triggered by the photochemical release of the ligands from their corresponding biologically inactive precursors (caged nucleotides; P3-1-(2-nitro)phenylethyl nucleotide). Binding of either ADP or ATP induced the appearance of small differential signals in the amide I band of the protein, sensitive to protein secondary structure, suggesting a subtle and localized change in protein conformation. Moreover, conformational changes associated with ATP hydrolysis were detected that differed markedly from those observed upon nucleotide binding. Both, high-amplitude absorbance changes and difference bands attributable to modifications in the interaction between oppositely charged residues were observed during ATP hydrolysis. Once this process had occurred, the protein relaxed to an ADP-like conformation. Our results suggest that the secondary structure as well as salt bridges of GroEL are modified during ATP hydrolysis, as compared with the ATP and ADP bound protein states.

ATP hydrolysis induces an intermediate conformational state in GroEL.

The conformational properties of the molecular chaperone GroEL in the presence of ATP, its non-hydrolyzable analog 5'-adenylimidodiphosphate (AMP-PNP), and ADP have been analyzed by differential scanning calorimetry (DSC), Fourier-transform infra-red (FT-IR) and fluorescence spectroscopy. Nucleotide binding to one ring promotes a decrease in the Tm value of the GroEL thermal transition that is reversed when both rings are filled with nucleotide, indicating that the sequential occupation of the two protein rings by these nucleotides has different effects on the GroEL thermal denaturation process. In addition, ATP induces a conformational change in GroEL characterized by (a) the appearance of a reversible low temperature endotherm in the DSC profiles of the protein, and (b) an enhanced binding of the hydrophobic probe 8-anilino-naphthalene-1-sulfonate (ANS), which strictly depends on ATP hydrolysis. The similar sensitivity to K+ of the temperature range where activation of the GroEL ATPase activity, the low temperature endotherm, and the increase of the ANS fluorescence are abserved strongly indicates the existence of a conformational state of GroEL during ATP hydrolysis, different from that generated on ADP or AMP-PNP binding. To achieve this intermediate conformation, GroEL mainly modifies its tertiary and quaternary structures, leading to an increased exposure of hydrophobic surfaces, with minor rearrangements of its secondary structure.

GroEL under heat-shock. Switching from a folding to a storing function.

Chaperonin GroEL from Escherichia coli, together with its cochaperonin GroES, are proteins involved in assisting the folding of polypeptides. GroEL is a tetradecamer composed of two heptameric rings, which enclose a cavity where folding takes place through multiple cycles of substrate and GroES binding and release. GroEL and GroES are also heat-shock proteins, their synthesis being increased during heat-shock conditions to help the cell coping with the thermal stress. Our results suggest that, as the temperature increases, GroEL decreases its protein folding activity and starts acting as a "protein store." The molecular basis of this behavior is the loss of inter-ring signaling, which slows down GroES liberation from GroEL and therefore the release of the unfolded protein from the GroEL cavity. This behavior is reversible, and after heat-shock, GroEL reverts to its normal function. This might have a physiological meaning, since under thermal stress conditions, it may be inefficient for the cell to fold thermounstable proteins that are prone to denaturation.

Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy.

We have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild-type hPAH was estimated to be 48% alpha-helix, 28% extended structures, 12% beta-turns and 12% non-structured conformations. The catalytic C-terminal domain (residues 112-452) holds most of the regular secondary structure elements, whereas the regulatory N-terminal domain (residues 2-110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L-Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N-terminal domain of the protein.

Permeabilization and fusion of uncharged lipid vesicles induced by the HIV-1 fusion peptide adopting an extended conformation: dose and sequence effects.

The peptide HIV(arg), corresponding to a sequence of 23 amino acid residues at the N-terminus of HIV-1 gp41 (LAV1a strain), has the capacity to destabilize negatively charged large unilamellar vesicles. As revealed by infrared spectroscopy, the peptide associated with those vesicles showed conformational polymorphism: in the absence of cations the main structure was a pore-forming alpha-helix, whereas in the presence of Ca2+ the conformation switched to a fusogenic, predominantly extended beta-type structure. Here we show that an extended structure can also be involved in electrically neutral vesicle destabilization induced by the HIV-1 fusion peptide when it binds the vesicle from the aqueous phase. In the absence of cations, neutral liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol (molar ratio 1:1:1) selected for an extended structure that became fusogenic in a dose-dependent fashion. At subfusogenic doses this structure caused the release of trapped 8-aminonaphtalene-1,3,6-trisulfonic acid sodium salt/p-xylenebis(pyridinium)bromide from liposomes, indicating the existence of a peptide-mediated membrane destabilizing process before and independent of the development of fusion. When compared to HIV(arg), the fusion activity of HIV(ala) (bearing the R22 --> A substitution) was reduced by 70%. Fusogenicity was completely abolished when a second substitution (V2 --> E) was included to generate HIV(ala-E2), a sequence representing the N-terminus of an inactive gp41. However, the three sequences associated with vesicles to the same extent, and the three adopted a similar extended structure in the membrane. Whereas 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene emission anisotropy was unaffected by the three peptides, DPH emission anisotropy in membranes was increased only by the fusogenic sequences. Taken together, our observations strongly argue that it is not an alpha-helical but an extended structure adopted by the HIV-1 fusion peptide what actively destabilizes cholesterol-containing, electrically neutral membranes. Moreover, membrane destabilization is modulated by the amino acid sequence in the extended structure. The effect displayed by the aforementioned V2 --> E substitution suggests that the fusion process described here could be reflecting a physiologically relevant phenomenon.

Effects of the inter-ring communication in GroEL structural and functional asymmetry.

The chaperonin GroEL consists of a double-ring structure that assists protein folding in the presence of GroES and ATP. Recent studies suggest that the 7-mer ring is the functional unit where protein folding takes place. Nevertheless, both GroEL rings are required to complete the reaction cycle through signals transmitted between the two rings. Electron microscopy, image processing, and biochemical analysis of GroEL, a single-ring mutant (SR1) and a inter-ring communication affected mutant (A126V), in the presence of ATP and adenylyl imidodiphosphate, have allowed the identification of a conformational change in the apical domains that is strictly dependent on the communication between the two GroEL rings. It is deduced from these results that the binding of nucleotide to both GroEL rings generates, as a consequence of the inter-ring communication, a functionally and structurally asymmetric particle. This asymmetric particle has a ring with a small conformational change in its apical domains and high affinity toward unfolded substrate and GroES, and the other ring has a larger conformational change in its apical domains and lower affinity toward substrate and GroES.

Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect of iron/catecholamine binding and phosphorylation.

The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% alpha-helix, 35% beta-extended structures (including beta-sheet), 14% beta-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in alpha-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.

Interaction of native and partially folded conformations of alpha-lactalbumin with lipid bilayers: characterization of two membrane-bound states.

alpha-Lactalbumin (alphaLA) can adopt two different membrane-bound states depending on the physical properties of the lipid bilayer, namely adsorbed and inserted. The latter, but not the adsorbed state, is able to disrupt the permeability barrier of the bilayer. The structure of both states is strongly affected by the conformational properties of the alphaLA conformer considered: as protein flexibility increases the helical content of the membrane-bound conformation decreases, especially in the adsorbed form. Moreover, the adsorbed and the inserted states of those conformers containing 3 or 4 disulfides can interconvert in response to changes in the physical properties of the host membrane.

Structural requirements for the association of native and partially folded conformations of alpha-lactalbumin with model membranes.

The effect of the structure and stability of several conformers of alpha-lactalbumin in aqueous solution on their association to negatively charged large unilamellar vesicles has been studied by circular dichroism, infrared spectroscopy, differential scanning calorimetry, and by content leakage experiments. Our results indicate that the affinity of alphaLA for negatively charged vesicles strongly depends on its conformational properties in solution. Analysis of the pH dependence of the interaction for the different conformers reveals that native-like, calcium-bound, ordered conformations become bilayer-associated through electrostatic forces. However, partially folded conformers are able to interact with negatively charged membranes at pHs higher than the protein isoelectric point, suggesting that hydrophobic interactions brought about by the exposure of hydorphobic residues at the protein surface are able to overcome the unfavorable electrostatic repulsion. Calorimetric and spectroscopic data in solution also indicate that substantial protein destabilization facilitates its subsequent membrane binding, and that the association process is favored for a set of conformers having significant secondary structure, but lacking native-like, stable tertiary structure. Aggregation of the unfolded alpha-lactalbumin molecules and burial of hydrophobic surfaces upon formation of ordered tertiary structure significantly reduce their membrane perturbing activity. These observations suggest that formation of a flexible strucutral intermediate of alpha-lactalbumin in solution is a prerequisite for its association with membranes.

Binding of molten globule-like conformations to lipid bilayers. Structure of native and partially folded alpha-lactalbumin bound to model membranes.

The effect of membrane binding on the structure and stability of several conformers of alpha-lactalbumin was studied by infrared spectroscopy, circular dichroism, and fluorescence spectroscopy. In solution, under experimental conditions where all conformers interact with negatively charged membranes, they show significant conformational differences. However, binding to negatively charged membranes, which causes considerable changes in the structure of these conformers, leads to a remarkably similar protein conformation. The membrane-associated conformations are characterized by 1) a high helical content, greater than any of those found in solution, 2) a lack of stable tertiary structure, and 3) the disappearance of their thermotropic transition. These observations indicate that association with negatively charged membranes induces a conformational change within alpha-lactalbumin to a flexible, molten globule-like state.