Structural study of the C2 domains of the classical PKC isoenzymes using infrared spectroscopy and two-dimensional infrared correlation spectroscopy

The secondary structure of the C2 domains of the classical PKC isoenzymes, alpha, betaII, and gamma, has been studied using infrared spectroscopy. Ca(2+) and phospholipids were used as protein ligands to study their differential effects on the isoenzymes and their influence on thermal protein denaturation. Whereas the structures of the three isoenzymes were similar in the absence of Ca(2+) and phospholipids at 25 degrees C, some differences were found upon heating in their presence, the C2 domain of the gamma-isoenzyme being better preserved from thermal denaturation than the domain from the alpha-isoenzyme and this, in turn, being better than that from the beta-isoenzyme. A two-dimensional correlation study of the denaturation of the three domains also showed differences between them. Synchronous 2D-IR correlation showed changes (increased aggregation of denaturated protein) occurring at 1616-19 cm(-1), and this was found in the three isoenzymes. On the other hand, the asynchronous 2D-IR correlation study of the domains in the absence of Ca(2+) showed that, in all cases, the aggregation of denaturated protein increased after changes in other structural components, an increase perhaps related with the hard-core role of the beta-sandwich in these proteins. The differences observed between the three C2 domains may be related with their physiological specialization and occurrence in different cell compartments and in different cells.     

Structure of beta-purothionin in membranes: a two-dimensional infrared correlation spectroscopy study

Two-dimensional infrared correlation spectroscopy has been used to investigate the structure of beta-purothionin, a small basic protein found in the endosperm of wheat seeds, in the absence and presence of dimyristoylphosphatidylglycerol (DMPG) membranes. To generate the two-dimensional synchronous and asynchronous maps, hydrogen-deuterium exchange of the protein amide protons has been used as an external perturbation. This method has allowed us to separate the different secondary structure elements and side chain contributions in the regions of amide I, II, and II' bands to determine that the relative order of deuteration of the beta-purothionin protons is as follows: turns, asparagines, and lysines > unordered structure and tyrosine > beta-sheet > alpha-helices and arginines. The results also indicate that the protein undergoes significant changes both in secondary structure and in deuteration in the presence of DMPG bilayers. The helical content of beta-purothionin is higher in the presence of the lipid, and the relative order of deuteration is as follows: lysines and arginines > asparagines and beta-sheet > unordered structure and alpha-helices. The inversion in the deuteration order of the arginine residues is assigned to a change of the degree of association of the protein in the membrane. In addition, the results reveal that the part of the protein containing the tyrosine residue interacts with the lipid membrane. Our results combined with those previously published suggest that the toxicity of beta-purothionin is more associated with the formation of functional channels in cell membranes rather than with a lytic phenomenon.     

Sequential events in ribonuclease A thermal unfolding characterized by two-dimensional infrared correlation spectroscopy

The conformational changes in the thermal denaturation of bovine pancreatic ribonuclease A was followed with infrared spectra and analyzed by second derivative and two-dimensional correlation techniques. By analyzing the sequential events in each transition stage, the results were consistent with a step-wise thermal denaturation mechanism in which the structural adjustment of the N-terminal and the opening of the central structure of the protein come before the main unfolding process. Non-native turns were found to form along with the unfolding of the native structures. The central region that is composed of some beta-sheet and alpha-helical structures was found to be the most stable part that might form the residual structure at high temperatures.     

Study on hydrogen bonds of carboxymethyl cellulose sodium film with two-dimensional correlation infrared spectroscopy

Carboxymethyl cellulose is widely used in many industrial aspects and also in laboratory due to its good biocompatibility. However, special researches on infrared especially aiming at the hydrogen bonds structure of carboxymethyl cellulose were relatively poor. We demonstrate here a full view of infrared spectroscopy in the temperature range of 40–220 °C, mainly aiming at the hydrogen bonds in the NaCMC film. The two important transition points was defined with DSC and together with Infrared analysis, that is, 100 °C corresponding to the complete loss of water molecules and 170 °C to the starting temperature point the O6H6 being oxidized. The series of IR spectra during heating from 40 to 220 °C was analyzed by the two-dimensional correlation method. We found that the water molecules bound with CO groups and OH groups. With the evaporating of water molecules, the hydrated CO groups gradually transited into non-hydrated CO groups. As the temperature continued to increase, the intrachain hydrogen bonds were weakened and transited into weak hydrogen bonds. When the temperature was higher than 170 °C, the O6H6 groups were gradually oxidized and thus the interchain hydrogen bonds formed between CH₂COONa groups and O6H6 were weakened. In summary, we defined the main sorts of hydrogen bonds in carboxymethyl cellulose and pictured the changes of the hydrogen bonds structure during heating process, which may provide for the further application in both industry aspects and laboratory use.     

Optical fingerprinting of peptides using two-dimensional infrared spectroscopy: proof of principle

We employ a particular form of two-dimensional infrared four-wave mixing (2DIR FWM) as a vibrational spectroscopic tool to quantify the amino acid content of a number of peptides. Vibrational features corresponding to ring modes of the aromatic groups of phenylalanine (Phe) and tyrosine (Tyr), as well as a methylene mode that is used as an internal reference, are identified. We show that the ratios of the integrated intensities, and the amplitudes, of the aromatic peaks of Phe and Tyr relative to the methylene integrated intensity, and amplitude, are proportional to the actual ratio of Phe and Tyr to CH₂ in the samples within a precision of ±12.5%. This precision is shown to be sufficient to use this form of 2DIR spectroscopy as a possible proteins fingerprinting tool.     

Further investigation on potassium-induced conformation transition of Nephila spidroin film with two-dimensional infrared correlation spectroscopy

We used two-dimensional (2D) correlation infrared spectroscopy to study further the potassium-induced conformation transition in Nephila spidroin films. It provided increased resolution and important new information on the sequence of events in the conformation transition process, showing that beta-sheet formed from the helical component before they formed from random coil. It also showed more evidence that formation of the 1691 cm(-1) (turn/bend) peak did not proceed with the same kinetics as the 1620 cm(-1) (antiparallel beta-sheet component) one, so we attribute the 1691 cm(-1) peak to turns which formed with different kinetics as the antiparallel beta-sheets. We present a single coherent and detailed hypothesis for the assembly and secondary structural transition of silk proteins in vivo and in vitro based on our findings and on evidence from other laboratories.     

Study on temperature-dependent changes in hydrogen bonds in cellulose Ibeta by infrared spectroscopy with perturbation-correlation moving-window two-dimensional correlation spectroscopy

Infrared (IR) spectra were measured for cellulose Ibeta prepared from the mantle of Halocynthia roretzi over a temperature range of 30-260 degrees C to explore the temperature-dependent changes in hydrogen bonds (H-bonds) in the crystal. Structural changes at the phase transition temperature of 220 degrees C are elucidated at the functional group level by perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy. The PCMW2D correlation spectra show that the intensities of bands arising from O3-H3...O5 and O2-H2...O6 intrachain H-bonds dramatically decrease at 220 degrees C, whereas the intensity changes of bands due to interchain H-bonds are not observed adequately. These results suggest that the phase transition is induced by the dissociation of the O3-H3...O5 and O2-H2...O6 intrachain H-bonds. However, the interchain H-bonds are not so much responsible for the transition directly.     

Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium cells has been studied using fluorescence correlation spectroscopy to gain a better understanding of the functioning of the domains and to assess the effect of initiation of chemotaxis on these domains in the cell. PH2-GFP was homogeneously distributed in vegetative as well as chemotaxing cells, whereas PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were very similar; however, PH10-GFP exhibited slower diffusion. Photon counting histogram statistics show that this slow diffusion was not due to aggregation. Diffusion of the three PH domains was affected to similar extents by intracellular heterogeneities in vegetative as well as chemotaxing cells. From the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP and PH-CRAC-GFP remained unchanged.     

Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes

We have used two-dimensional infrared correlation spectroscopy (2D-IR) to study the interaction and conformation of cytochrome c in the presence of a binary phospholipid mixture composed of a zwitterionic perdeuterated phospholipid and a negatively-charged one. The influence of the main temperature phase transition of the phospholipid model membranes on the conformation of cytochrome c has been evaluated by monitoring both the Amide I' band of the protein and the CH(2) and CD(2) stretching bands of the phospholipids. Synchronous 2D-IR analysis has been used to determine the different secondary structure components of cytochrome c which are involved in the specific interaction with the phospholipids, revealing the existence of a specific interaction between the protein with cardiolipin-containing vesicles but not with phosphatidic acid-containing ones. Interestingly, 2D-IR is capable of showing the existence of significant changes in the protein conformation at the same time that the phospholipid transition occurs. In summary, 2D-IR revealed an important effect of the phospholipid phase transition of cardiolipin on the secondary structure of oxidized cytochrome c but not to either reduced cytochrome c or in the presence of phosphatidic acid, demonstrating the existence of specific intermolecular interactions between cardiolipin and cytochrome c.     

A Fourier transform infrared spectroscopy study of cell membrane domain modifications induced by docosahexaenoic acid

Background

Detergent resistant membranes (DRMs) are a useful model system for the in vitro characterization of cell membrane domains. Indeed, DRMs provide a simple model to study the mechanisms underlying several key cell processes based on the interplay between specific cell membrane domains on one hand, and specific proteins and/or lipids on the other. Considering therefore their biological relevance, the development of methods capable to provide information on the composition and structure of membrane domains and to detect their modifications is highly desirable. In particular, Fourier transform infrared (FTIR) spectroscopy is a vibrational tool widely used for the study not only of isolated and purified biomolecules but also of complex biological systems, including intact cells and tissues. One of the main advantages of this non-invasive approach is that it allows obtaining a molecular fingerprint of the sample under investigation in a rapid and label-free way.

Methods

Here we present an FTIR characterization of DRM fractions purified from the human breast cancer cells MCF-7, before and after treatment with the omega 3 fatty acid docosahexaenoic acid (DHA), which was found to promote membrane microdomain reorganization.

Results and Conclusions

We will show that FTIR spectroscopy coupled with multivariate analysis enables to monitor changes in the composition of DRMs, induced in particular by the incorporation of DHA in cell membrane phospholipids.

General significance

This study paves the way for a new label-free characterization of specific membrane domains within intact cells, which could provide complementary information to the fluorescence approaches presently used.     

Structural determination of the vasoactive intestinal peptide by two-dimensional H-NMR spectroscopy

The structure of the vasoactive intestinal peptide 1-28 in 40% 2,2,2-trifluoroethanol was investigated by two-dimensional 1H-nmr spectroscopy. All 1H resonances, except the gamma, delta, and epsilon protons of the lysine residues, could be sequentially assigned. Numerous intraresidual as well as short-range interresidual nuclear Overhauser effect spectroscopy connectivities were observed. Using a variable-target function minimization, a molecular model consisting of two helical stretches involving residues 7-15 and 19-27 connected by a region of undefined structure was calculated. The existence of an undefined structure between residues 16 and 18 confers mobility to the peptide molecule.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

Structural characterization of the catalytic domain of the human 5-lipoxygenase enzyme

Leukotrienes are inflammatory mediators involved in several diseases. The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Little structural information is available regarding 5-lipoxygenase. In this study, we found that the primary structure of the catalytic domain of human 5-lipoxygenase is similar to that of the rabbit 15-lipoxygenase. This similarity allowed the development of a theoretical model of the tertiary structure of the 5-lipoxygenase catalytic domain, using the resolved structure of rabbit 15-lipoxygenase as a template. This model was used in conjunction with primary and secondary structural information to investigate putative nucleotide binding sites, a MAPKAP kinase 2 phosphorylation site, and a Src homology 3 binding site on the 5-lipoxygenase protein, further. Results indicate that the putative nucleotide binding sites are spatially distinct, with one on the -barrel domain and the other(s) on the catalytic domain. The MAPKAP kinase 2 phosphorylation site involves a four amino acid insertion in mammalian 5-lipoxygenases that significantly alters molecular structure. This target for post-translational modification is both common and unique to 5-lipoxygenases. The Src homology 3 binding site, found in all lipoxygenases, appears to lack the characteristic left-handed type II helix structure of known Src homology 3 binding sites. These results, which highlight the unique nature of the MAPKAP kinase site, underscore the utility of structural information in the analysis of protein function. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0076-y.Electronic Supplementary Material available.     

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.     

Structural polymorphism of telomeres studied by photon correlation spectroscopy

Photon Correlation Spectroscopy (PCS) was used to study the dynamics and structure of Tetrahymena telomeric sequence d(5'-TGGGGT-3')4. Two different modes were observed, corresponding to the following structures: intermolecular (tetramolecular) G-quadruplex and intramolecular (monomeric) G-quartet. Experimental values of translational diffusion coefficients DT were obtained for each structural form. The value of DT for the monomer equals to 1.4 x 10(6) (cm2/s), while for the tetramolecular structure, to 0.8 x 10(6) (cm2/s). The relative weight concentrations of these two forms were analyzed versus the concentration of NaCl varied from 10 mM to 500 mM. The values of experimentally determined diffusion coefficients were compared with those calculated assuming the "bead model" and with the atomic coordinates from the NMR and X-ray crystallographic data. For both structures the experimental and calculated values of DT were in reasonable agreement. In the entire NaCl concentration range studied, the contribution of the relative weight concentration of the monomeric telomere form changed from 85% for 10 mM NaCl to 60% for 500 mM NaCl.     

Temperature-dependent changes in hydrogen bonds in cellulose Ialpha studied by infrared spectroscopy in combination with perturbation-correlation moving-window two-dimensional correlation spectroscopy: comparison with cellulose Ibeta

Our recent IR study demonstrated that hydrogen-bond structure in cellulose Ibeta drastically changes around 220 degrees C (Watanabe et al. Biomacromolecules 2006, 7, 3164). In the present study, temperature-dependent IR spectra of cellulose Ialpha from 30 to 260 degrees C were analyzed by use of perturbation-correlation moving-window two-dimensional correlation spectroscopy. It was observed that as in the case of cellulose Ibeta abrupt changes in the hydrogen-bond structure occur around 220 degrees C in cellulose Ialpha. It was also revealed that although weakly hydrogen-bonded OH groups in Ibeta are stable below 230 degrees C thermal oxidation of those in Ialpha is accelerated around 220 degrees C. In this way, the present study has clarified a difference between the thermal behavior of Ialpha and that of Ibeta at the functional group level. Our result suggests that the drastic change in the hydrogen-bond structure around 220 degrees C makes cellulose Ialpha much more unstable than Ibeta.     

Time-resolved infrared spectroscopy in the study of photosynthetic systems

Time-resolved (TR) infrared (IR) spectroscopy in the nanosecond to second timescale has been extensively used, in the last 30 years, in the study of photosynthetic systems. Interesting results have also been obtained at lower time resolution (minutes or even hours). In this review, we first describe the used techniques—dispersive IR, laser diode IR, rapid-scan Fourier transform (FT)IR, step-scan FTIR—underlying the advantages and disadvantages of each of them. Then, the main TR-IR results obtained so far in the investigation of photosynthetic reactions (in reaction centers, in light-harvesting systems, but also in entire membranes or even in living organisms) are presented. Finally, after the general conclusions, the perspectives in the field of TR-IR applied to photosynthesis are described.     

Molecular conformation of gonadoliberin using two-dimensional NMR spectroscopy

Complete resonance assignments of the proton NMR spectrum of gonadoliberin (in its native amide and free acid forms) have been obtained using two-dimensional nuclear magnetic resonance spectroscopy under three different environmental conditions, namely, dimethyl sulphoxide solution, aqueous solution and lipid-bound form in model membranes. The proton chemical shifts in the three cases have been compared to derive information about inherent conformational characteristics of the molecule. It has been inferred that the molecule possesses no short-range or long-range order under any of the three solvent conditions. However, there is a nonspecific increase in the linewidths when gonadoliberin is bound to model membranes, indicating a reduced internal motion in the molecule due to lipid-peptide interactions.     

Assignments and structure determination of the catalytic domain of human fibroblast collagenase using 3D double and triple resonance NMR spectroscopy

We report here the backbone 1HN, 15N, 13C, 13CO, and 1H NMR assignmentsfor the catalytic domain of human fibroblast collagenase (HFC). Three independentassignment pathways (matching 1H, 13C, and 13CO resonances) were used to establishsequential connections. The connections using 13C resonances were obtained fromHNCOCA and HNCA experiments; 13CO connections were obtained from HNCO andHNCACO experiments. The sequential proton assignment pathway was established from a 3D(1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement withrecent X-ray structures of human fibroblast collagenase and human neutrophil collagenase aswell as similar to recent NMR structures of a highly homologous protein, stromelysin. Allthree helices were located; a five-stranded -sheet (four parallel strands, one antiparallelstrand) was also determined. -Sheet regions were identified by cross-stranddN and dNN connections and by strong intraresidue dN correlations, and were corroborated byobserving slow amide proton exchange. Chemical shift changes in a selectively 15N-labeledsample suggest that substantial structural changes occur in the active site cleft on the bindingof an inhibitor.     

Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes

We have used two-dimensional infrared correlation spectroscopy (2D-IR) to study the interaction and conformation of cytochrome c in the presence of a binary phospholipid mixture composed of a zwitterionic perdeuterated phospholipid and a negatively-charged one. The influence of the main temperature phase transition of the phospholipid model membranes on the conformation of cytochrome c has been evaluated by monitoring both the Amide I′ band of the protein and the CH₂ and CD₂ stretching bands of the phospholipids. Synchronous 2D-IR analysis has been used to determine the different secondary structure components of cytochrome c which are involved in the specific interaction with the phospholipids, revealing the existence of a specific interaction between the protein with cardiolipin-containing vesicles but not with phosphatidic acid-containing ones. Interestingly, 2D-IR is capable of showing the existence of significant changes in the protein conformation at the same time that the phospholipid transition occurs. In summary, 2D-IR revealed an important effect of the phospholipid phase transition of cardiolipin on the secondary structure of oxidized cytochrome c but not to either reduced cytochrome c or in the presence of phosphatidic acid, demonstrating the existence of specific intermolecular interactions between cardiolipin and cytochrome c.