Production and initial structural characterization of the TM4TM5 helix-loop-helix domain of the translocator protein.

Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helix-helix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C-terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far-UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n-dodecyl-ß-d-maltoside, n-octyl-ß-d-glucoside, n-dodecylphosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5-DHPC complex.

Imaging capabilities of synchrotron infrared microspectroscopy.

It has become increasingly clear that synchrotron infrared micro-spectroscopy is an extremely valuable analysis tool when determining the chemical composition of biological and biomedical samples, at the diffraction-limited spatial resolution. Highly resolved infrared micro-spectroscopy, together with the high signal-to-noise level of the recorded spectra, is essential in generating chemical and statistical (multivariate) images. This is illustrated in the case of individual cell and hair section studies. Unprecedented chemical images of lipid distribution and secondary structure relative concentration have been achieved using the synchrotron source. A comparison with a Focal plane Array imaging system, on the same hair section, shows that, despite the fast imaging processing and improved quality achieved with the focal plane array detectors, spectral quality is markedly superior in the case of the synchrotron source. It is clear that the two approaches could be very complementary if combined on the same sample area, in a synchrotron facility.

Two temporal stages of oligodendroglial response to excitotoxic lesion in the gray matter of the adult rat brain.

Excitotoxic lesions in the gray matter induce profuse demyelination of passage and afferent fibers in areas of neuronal loss, independent of Wallerian degeneration. The time course of this phenomenon, which extends over weeks after the excitotoxin injection, suggests that demyelination is not related only to a direct effect of the toxin. In order to define mechanisms at work, a parallel study of myelin and oligodendrocytes was carried out following kainate injections into the adult rat thalamus. Within the 1st day postlesion, myelin alteration appeared throughout the area exhibiting neuronal loss, while the number of oligodendrocytes fell by 45%. No apoptotic oligodendrocytes were identified at that time. Over the following 2 days, there was no further loss of myelin and oligodendrocytes, but there was an increase in the number of oligodendrocytes displaying typical signs of apoptosis as revealed with TUNEL-end-labeled nuclei, Hoechst-labeled condensed chromatin bodies, or bax immunoreactivity. This resulted in a second, progressive loss of both myelin and oligodendrocytes leading to their almost complete disappearance 2 weeks postlesion. These results demonstrate two temporal stages of oligodendroglial cell death. The excitotoxin injection resulted in the rapid destruction of a first oligodendroglial population, most probably by necrosis. A second population died in a delayed manner from apoptosis. This second wave of death coincided with an activated microglia/macrophage invasion of the lesion, suggesting that delayed oligodendroglial death results from toxic microglia/macrophage effects. In addition, the longest surviving oligodendrocytes were located next to reactive astrocytes, suggesting the existence of trophic interactions between these two glial populations.

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex. The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.

A gradual disruption of tight side-chain packing: 2D 1H-NMR characterization of acid-induced unfolding of CHABII.

Little is known about the mechanism of the transition between native proteins and partially folded intermediates. Complete assignments of 2D 1H-NOESY spectra of CHABII at 5 degrees C, pH 6.3, 5.5, 4.6 and 4.0, reveal that lowering of pH results in an extensive but gradual disappearance of NOEs, implying a gradual disruption of tight side-chain packing. Moreover, a tertiary packing core is identified at 5 degrees C and pH 4.0, characterized by persistent long-range NOEs. Thus, we suggest that severe disruption of tight side-chain packing of CHABII can occur at a stage where its secondary structure and tertiary topology remain highly native-like.

Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy.

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13C(alpha) 13CO, 1H(alpha), and 13Cbeta NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple (1H-13C-15N) and double resonance (1H-15N or 1H-13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3(J)H(N)H(alpha) and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro 151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13C(alpha) chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.

Highly resolved chemical imaging of living cells by using synchrotron infrared microspectrometry.

Using synchrotron radiation as an ultra-bright infrared source, we have been able to map the distributions of functional groups such as proteins, lipids, and nucleic acids inside a single living cell with a spatial resolution of a few microns. In particular, we have mapped the changes in the lipid and protein distributions in both the final stages of cell division and also during necrosis.

Chemical imaging of nucleic acids, proteins and lipids of a single living cell. Application of synchrotron infrared microspectrometry in cell biology.

Hybridoma B-cells have been used as models to evaluate the performance of synchrotron infrared microscopy to obtain chemical images of a single living cell. Chemical mapping of nucleic acids, proteins and lipids at a resolution of a few microns, close to the diffraction limit in the mid-infrared region are shown.

NMR solution structure of a two-disulfide derivative of charybdotoxin: structural evidence for conservation of scorpion toxin alpha/beta motif and its hydrophobic side chain packing.

The alpha/beta scorpion fold consisting of a short alpha-helix and beta-sheet is a structural motif common to scorpion toxins, insect defensins, and plant gamma-thionins that invariably contains three disulfides. CHABII is a two-disulfide derivative of the scorpion toxin charybdotoxin (ChTX), chemically synthesized by inserting two L-alpha-aminobutyric acids in place of the two half-cystine residues involved in the disulfide 13-33. This disulfide is one of the two disulfides which connect the alpha-helix to the beta-sheet. The solution structure of CHABII was determined at pH 6.3 and 5 degrees C using 2D NMR and simulated annealing from 513 distance and 46 dihedral angle constraints. The NMR structure of CHABII is well-defined as judged from the low value of the averaged backbone rms deviation between the 30 lowest energy structures and the energy-minimized mean structure ((rmsd) = 0.65 A for the entire sequence and 0.48 A for the segment 3-36). Analysis and comparison of the solution structures of CHABII and ChTX lead to the following conclusions: (i) the fold of CHABII is similar to that of ChTX as indicated by the low value of the averaged backbone atomic rms deviation between the 10 lowest energy solution structures of the two proteins (1.44 A); (ii) the packing of the hydrophobic core is well-preserved, underlying the critical structural role of the hydrophobic interactions even for such a small and cysteine-rich protein as ChTX.

On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures.

BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 +/- 1.0 A. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.

Flexibility in the second half-site sequence recognised by the c-Myb R2 domain--in vitro and in vivo analysis.

The oncoprotein c-Myb is a transcription factor that recognises its specific target sequences through two subdomains. The R3-domain binds the first half-site, YAAC, and plays a dominant role in sequence recognition, while the homologous R2-domain interacts with a more loosely defined sequence in the second half-site. The difficulty in precisely defining a preferred second half-site sequence might reflect the flexible nature of R2 which only attains its fully folded structure upon binding to DNA, a process that might allow the protein to adapt to different half-site sequences. Here we report that shifting the most conserved base in the second half-site, the G6, into position 5 resulted only in a minor reduction of complex stability in vitro. From an analysis of a series of second half-site variants by EMSA and DMS-interference, we conclude that the preferred recognition sequence should be revised to read [YAACNG or YAACGN]. Modeling the structure of c-Myb R2R3 in complex with a GT half-site variant revealed specific interactions with G5. When second half-site variants were tested in vivo using a sensitive yeast effector-reporter system, both the TG and GT half-site variants were functional mediating c-Myb-dependent transactivation. Unexpectedly, we observed large differences between the best second half-site variants at low levels of c-Myb-effector, the GG variant being five- to fifteen-fold more active in vivo than the single-G half-sites, the GH or HG variants.

DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.

Secondary structure of the DNA-binding domain of the c-Myb oncoprotein in solution. A multidimensional double and triple heteronuclear NMR study.

The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix-turn-helix (HTH) motif. As a first step towards determination of the three-dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly 15N-labeled and 13C, 15N double-labeled R2R3 and, a selectively 15N-enriched sample on all lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 1H, 15N and 13C" atoms and determine the secondary structure of R2R3 in solution. The R3 repeat is composed of three helices (residues 62-75, 78-85 and 91-100) while for the R2 repeat only two helices are found (residues 10-23 and 28-34). The remaining C-terminal part of the R2 repeat, predicted to be helical and part of the HTH motif, undergoes intermediate conformational exchange processes. Stabilization of this segment might occur upon binding to DNA.

Preliminary assignments of the aromatic and some methyl group resonances of the 1H-NMR spectrum of the oxidized form of uteroglobin. Application to the interaction of oxidized uteroglobin with progesterone.

Two-dimensional NMR methods have been used to assign aromatic and methyl group resonances in the 1H-NMR spectrum of oxidized uteroglobin. Assignments to specific amino acids are based on X-ray-determined structures of two crystal forms (C222(1) and P2(1] and on an energy-minimized X-ray structure of the C222(1) form of uteroglobin. These preliminary assignments are sufficient to probe the interaction of oxidized uteroglobin with progesterone in solution. The protein global structure is unmodified but some direct or indirect conformational changes are induced in the H1H4(H1'H4') pockets and close to Phe28 by progesterone.

Two-dimensional nuclear Overhauser enhancement investigation of the solution structure and dynamics of the DNA octamer [d(GGTATACC)]2.

The resonances of nearly all 70 of the non-exchangeable protons of the duplex [d(GGTATACC)]2 in aqueous solution are assigned by proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra obtained in pure absorption phase at 500 MHz. Experimental and theoretical 2D NOE spectra are compared at each mixing time (100, 175, 250 and 400 ms) using two B-DNA structures: a standard B-form and an energy-minimized form. The GG and CC ends of the octamer duplex are well represented by the regular B-DNA structure. But large discrepancies from these models are observed for the 'TATA' box. All 2D NOE data are consistent with nanosecond correlation times, as indicated by non-selective proton spin-lattice relaxation times, but small variations in the correlation time are observed, suggesting that there are some local differences in mobility within the octamer duplex structure in solution.