Investigation of secondary and tertiary structural changes of cytochrome c in complexes with anionic lipids using amide hydrogen exchange measurements: an FTIR study

The structure of cytochrome c bound to anionic lipid membranes composed of dimyristoyl, dipalmitoyl, or dioleoyl phosphatidylglycerols, or of bovine heart cardiolipin, has been investigated by Fourier transform infrared spectroscopy. Only small changes in secondary structure, as registered by the amide I band of cytochrome c, were observed upon binding at temperatures below that of denaturation of the protein, and these were not coupled to the thermotropic phase transitions of the lipid. The denaturation temperature of the protein decreased by approximately 25-30 degrees upon binding, in a progression which correlated with that of the lipid phase transition temperatures, being approximately 7 degrees lower for complexes with dioleoyl than with dipalmitoyl phosphatidylglycerol. Large changes in the amide proton exchange characteristics, as monitored by the spectral shifts in the amide I band of the protein in D2O, were observed on binding cytochrome c to the lipid membranes. For the slowly exchanging population, the amide deuteration rates of the free protein were nearly independent of temperature, whereas those of the bound protein increased by up to two orders of magnitude over the temperature range from 10 to 40 degrees C. In addition, the extent of exchange differed between the bound and unbound protein. A structural transition in the bound protein was detected as a discontinuous step in Arrhenius plots of the deuterium exchange rates which occurred at a temperature in the region of 22 to 29 degrees C, depending on the lipid, far below that of denaturation. The temperature of this transition was determined by the physical state of the lipid, being 7 degrees lower for the lipids in the fluid state than for those in the gel state, and, for complexes with dimyristoyl phosphatidylglycerol, occurred at an intermediate temperature, being controlled by the lipid chain-melting transition at 27-28 degrees C. These results provide evidence for a coupling of the tertiary structure of the membrane-bound protein with the physical state of the membrane lipids.     

Synthesis and evaluation of deglycobleomycin A2 analogues containing a tertiary N-methyl amide and simple ester replacement for the L-histidine secondary amide: direct functional characterization of the requirement for secondary amide metal complexation

The synthesis and comparative examination of 3-5, analogues of deglycobleomycin A2 (2) which address the inferred importance of the L-histidine secondary amide directly, are detailed. The agent 3 lacks only the L-histidine beta-hydroxy group of deglycobleomycin A2 and the corresponding agents 4 and 5 incorporate a tertiary N-methyl amide and simple ester in place of the L-histidine secondary amide. The DNA cleavage properties of 3 proved essentially indistinguishable from those of deglycobleomycin A2 (2) confirming that the distinctions between bleomycin A2 (1) and deglycobleomycin (2) are due to the removal of the disaccharide and not the introduction of the L-histidine free beta-hydroxy group. The agents 4 and 5 containing a tertiary N-methyl amide and ester in place of the L-histidine secondary amide were found to cleave duplex DNA but to do so in a nonsequence selective fashion with a substantially reduced efficiency and a diminished double to single strand cleavage ratio that are only slightly greater than that of free iron itself. These latter observations establish the functional requirement for the L-histidine secondary amide and are consistent with the proposals that the L-histidine deprotonated secondary amide is required for functional metal chelation and activity.     

The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins

Proteins can be denatured by pressures of a few hundred MPa. This finding apparently contradicts the most widely used model of protein stability, where the formation of a hydrophobic core drives protein folding. The pressure denaturation puzzle is resolved by focusing on the pressure-dependent transfer of water into the protein interior, in contrast to the transfer of nonpolar residues into water, the approach commonly taken in models of protein unfolding. Pressure denaturation of proteins can then be explained by the pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. Activation volumes for hydrophobic contributions to protein folding and unfolding kinetics are positive. Clathrate hydrates are predicted to form by virtually the same mechanism that drives pressure denaturation of proteins.     

Time-dependent changes in the response of cartilage to static compression suggest interstitial pH is not the only signaling mechanism

The goal of the present study was to reexamine the role of interstitial pH in regulating the biosynthetic rate in cartilage tissue by addressing two research questions: (a) Do small, short-term changes in interstitial pH, induced independently by two different mechanisms (namely, by controlling the pH of the medium or by mechanical compression), result in biosynthetic rates commensurate with those expected from the "natural" relationship between interstitial pH and biosynthesis? and (b) Are the effects of changes in the pH of the medium or in compression the same for short-term (14-hour) and long-term (60-hour) exposures? Biosynthetic rates were estimated from incorporation of sulfate and proline into explants of bovine epiphyseal cartilage during the final 14 hours of culture. These rates decreased with decreasing pH of the medium, with increasing compression, and with decreasing native glycosaminoglycan content; or, expressed in terms of interstitial pH, acidification induced by compression or by lowering the pH of the medium resulted in a decreased biosynthetic rate, whereas interstitial acidification effected by increasing glycosaminoglycan content enhanced it. When the time for which tissue was exposed to changes in the pH of the medium was increased from 14 to 60 hours, the relationship between the biosynthetic rate and the pH remained constant whereas the relationship between the biosynthetic rate and compression was reversed. These data suggest that the transduction mechanisms underlying the response to pH of the medium and compression differ and that some adaptation or stimulation by modest levels of compression can occur with longer exposures. Interstitial pH is not the sole determinant of biosynthesis, and it cannot really account for the long-term response of cartilage tissue to static compression.     

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron PI.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.     

Inter-helical interactions in the leucine zipper coiled coil dimer: pH and salt dependence of coupling energy between charged amino acids

We have investigated the physical nature of the observed coupling energy (Delta Delta DeltaGint) between the charged side-chains of the three inter-helical g<-->e' (i, i'+5) pairs (E<-->R, E<-->K, and E<-->E) in the leucine zipper coiled coil dimer. Circular dichroism (CD) spectroscopy measured the thermal stability of eight proteins derived from the basic region leucine zipper domain of chicken VBP, the mammalian TEF at seven pHs and three KCl concentrations. Data from these proteins were used to construct double mutant alanine thermodynamic cycles and determine coupling energies (Delta Delta DeltaGint) for the three g<-->e' pairs. The attractive E<-->R coupling energy of -0.6 kcal mol-1 at low salt decreases to -0.2 kcal mol-1 at high salt. The E<-->K coupling energy of -0.5 kcal mol-1 at low salt decreases to -0.1 kcal mol-1 at high salt. The repulsive E<-->E coupling energy of +0.8 kcal mol-1 at low salt drops to +0.4 at high salt. Reducing the pH to 2.2 halved the attractive coupling energy for the E<-->R and E<-->K pairs while abolishing the repulsion of the E<-->E pair. 13C NMR of a protein selectively labeled with [13Cdelta]glutamate that contained three E<-->R and one R<-->E pair identified four glutamates shifted upfield. We suggest that this is due to electronic perturbation of glutamates in inter-helical E<-->R interactions. Taken together, these data indicate that the E<-->R coupling energy of -0.5 kcal mol-1 at pH 7.4 and 150 mM KCl has an electrostatic component.     

Role of main-chain electrostatics, hydrophobic effect and side-chain conformational entropy in determining the secondary structure of proteins

The physiochemical bases of amino acid preferences for alpha-helical, beta-strand, and other main-chain conformational states in proteins is controversial. Hydrophobic effect, side-chain conformational entropy, steric factors, and main-chain electrostatic interactions have all been advanced as the dominant physical factors which determine these preferences. Many attempts to resolve the controversy have focused on small model systems. The disadvantage of such systems is that the amino acids in small molecules are largely exposed to the solvent. In proteins, however, the amino acids are in contact with the solvent to a different degree, causing a large variability of strengths of all interactions. The estimates of mean strengths of interactions in the actual protein environment are therefore essential to resolve the controversy. In this work the experimental protein structures are used to estimate the mean strengths of various interactions in proteins. The free energy contributions of the interactions are implemented into the Lifson-Roig theory to calculate the helix and strand free energy profiles. From the profiles the secondary structures of proteins and peptides are predicted using simple rules. The role of hydrophobic effect, side-chain conformational entropy, and main-chain electrostatic interactions in determining the secondary structure of proteins is assessed from the abilities of different models, describing stability of secondary structures, to correctly predict alpha-helices, beta-strands and coil in 130 proteins. The three-state accuracy of the model, which contains only the free energy terms due to the main-chain electrostatics with 40 coefficients, is 68.7%. This accuracy is approaching to the accuracy of currently the best secondary structure prediction algorithm based on neural networks (72%); however, many thousands of parameters have to be optimized during the training of the neural networks to reach this level of accuracy. The correlation coefficient between the calculated and the experimental helix contents of 37 alanine based peptides is 0.91. If the hydrophobic and the side-chain conformational entropy terms are included into the helix-coil transition parameters, the accuracy of the algorithm does not improve significantly. However, if the main-chain electrostatic interactions are excluded from the helix-coil and strand-coil transition parameters, the accuracy of the algorithm reaches only 59.5%. These results support the dominant role of the short-range main-chain electrostatics in determining the secondary structure of proteins and peptides. The role of the hydrophobic effect and the side-chain conformational entropy is small.     

Conserved sequence preference in DNA binding among recombination proteins: an effect of ssDNA secondary structure

Repetitive sequences have been proposed to be recombinogenic elements in eukaryotic chromosomes. We tested whether dinucleotide repeats sequences are preferential sites for recombination because of their high affinity for recombination enzymes. We compared the kinetics of the binding of the scRad51, hsRad51 and ecRecA proteins to oligonucleotides with repeats of dinucleotides GT, CA, CT, GA, GC or AT. Since secondary structures in single-stranded DNA (ssDNA) act as a barrier to complete binding we measured whether these oligonucleotides are able to form stable secondary structures. We show that the preferential binding of recombination proteins is conserved among the three proteins and is influenced mainly by secondary structures in ssDNA.     

Kinetics of the interactions of a dicarboxylic porphyrin with unilamellar lipidic vesicles: interplay between bilayer thickness and pH in rate control

The transfer of a dicarboxylic porphyrin from phosphatidylcholine fluid-phase unilamellar vesicles towards albumin is studied focusing on bilayer thickness and pH effects. The kinetics of this process yield the rate constants for the porphyrin flip-flop from the inner to the outer hemileaflet and its exit towards aqueous medium. Phospholipids with monounsaturated 14-22 carbon chains are used. Interplay between bilayer thickness and pH for the control of the rate constants is observed. This results in the amplification, at physiological pH, of the effect of membrane thickness on the flip-flop and exit rates as compared to pH 8.5 and 6.5. These data are explained by the degree of porphyrin burying within the bilayer resulting from a compromise between favorable hydrophobic interactions with the hydrocarbon phase and unfavorable penetration of the polar carboxylic chains. The balance between the two effects depends particularly on the neutralization of one carboxylic chain. Considering the bilayer hydrophobicity profile and the porphyrin size, the optimization of hydrophobic interactions appears dependent on the bilayer thickness. The flip-flop and the exit are governed by neutralization and deprotonation of the carboxylic chains, respectively, the rate of these proton exchanges being dependent on the porphyrin location within the bilayer.     

NMR spectroscopy of alpha-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of alpha-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved 1H NMR spectrum is observed for alpha-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-crystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of alpha A-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic alpha-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'substrate' protein. The conformation of 'substrate' proteins when they interact with alpha-crystallin has been probed by 1H NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the 1H and 31P NMR spectra of alpha-crystallin in the presence of increasing concentrations of urea, it is proposed that alpha-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of alpha-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with 'substrate' proteins during the chaperone action.     

The great escape: structure and function of the autotransporter proteins

The autotransporters, a family of secreted proteins from Gram-negative bacteria, possess an overall unifying structure comprising three functional domains: the amino-terminal leader sequence, the secreted mature protein (passenger domain) and a carboxy-terminal (beta-) domain that forms a beta-barrel pore to allow secretion of the passenger protein. Members of this family have been implicated as important or putative virulence factors in many Gram-negative pathogens.     

Water molecules in DNA recognition II: a molecular dynamics view of the structure and hydration of the trp operator

The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2 corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(+/-19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(+/-11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(+/-4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3' and O4' and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA.     

Clock-spiking cells not only in the eye of the fly, but also in the antenna!

Progressive pseudorheumatoid dysplasia (spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDTPA) or progressive pseudorheumatoid arthropathy of childhood (PPAC) (MIM 208.230)) is an autosomal recessively inherited skeletal dysplasia with changes in the spine similar to spondyloepiphyseal dysplasia tarda. The disease begins mostly between the ages of 2 to 8 years with progressive joint stiffness and pain, soft tissue swelling and deformities of multiple joints including the proximal interphalangeal joints of the hands. We describe a patient where a symmetric rhizomelic shortening of the extremities and a bilateral severe pes equinovarus was present at birth.     

The distribution of direct interactions in the spatial structures of globular proteins

The hydrogen bond distributions in 123 protein structures with the atom coordinates established at a resolution of less than 2 A were analyzed. The peculiarities of hydrogen bond distributions with respect to the lengths and remoteness of contacting residues in the primary structure were established. A hierarchy of H-bond energy distribution in the spatial structure of protein globules was demonstrated. The role of hydrogen bonds in the formation of domain structure and their special properties in proteins with different types of secondary structure are discussed.