Hydration of biological molecules: lipids versus nucleic acids.

We used FTIR spectroscopy to comparatively study the hydration of films prepared from nucleic acids (DNA and double-stranded RNA) and lipids (phosphatidylcholines and phosphatidylethanolamines chosen as the most abundant ones) at room temperature by varying the ambient relative humidity in terms of solvent-induced structural changes. The nucleic acids and phospholipids both display examples of polymorphism on the one hand and structural conservatism on the other; even closely related representatives behave differently in this respect. DNA undergoes a hydration-driven A-B conformational transition, but RNA maintains an A-like structure independently of the water activity. Similarly, a main transition between the solid and liquid-crystalline phases can be induced lyotropically in certain phosphatidylcholines, while their phosphatidylethanolamine counterparts do not exhibit chain melting under the same conditions. A principal difference concerning the structural changes that occur in the studied biomolecules is given by the relevant water-substrate stoichiometries. These are rather high in DNA and often low in phospholipids, suggesting different mechanisms of action of the hydration water that appears to induce structural changes on global- and local-mode levels, respectively.

Solution structure of the dodecamer d-(CATGGGCC-CATG)2 is B-DNA. Experimental and molecular dynamics study.

The DNA duplex d-(CATGGGCCCATG)2 has been studied in solution by FTIR, NMR and CD. The experimental approaches have been complemented by series of large-scale unrestrained molecular dynamics simulation with explicit inclusion of solvent and counterions. Typical proton-proton distances extracted from the NMR spectra and the CD spectra are completely in agreement with slightly modified B-DNA. By molecular dynamics simulation, starting from A-type sugar pucker, a spontaneous repuckering to B-type sugar pucker was observed. Both experimental and theoretical approaches suggest for the dodecamer d-(CATGGGCCCATG)2 under solution conditions puckering of all 2'-deoxyribose residues in the south conformation (mostly C2'-endo) and can exclude significant population of sugars in the north conformation (C3'-endo). NMR, FTIR and CD data are in agreement with a B-form of the dodecamer in solution. Furthermore, the duplex shows a cooperative B-A transition in solution induced by addition of trifluorethanol. This contrasts a recently published crystal structure of the same oligonucleotide found as an intermediate between B- and A-DNA where 23 out of 24 sugar residues were reported to adopt the north (N-type) conformation (C3'-endo) like in A-DNA (Ng, H. L., Kopka, M. L. and Dickerson, R. E., Proc. Natl. Acad. Sci. U S A 97, 2035-2039 (2000)). The simulated structures resemble standard B-DNA. They nevertheless show a moderate shift towards A-type stacking similar to that seen in the crystal, despite the striking difference in sugar puckers between the MD and X-ray structures. This is in agreement with preceding MD reports noticing special stacking features of G-tracts exhibiting a tendency towards the A-type stacking supported by the CD spectra also reflecting the G-tract stacking. MD simulations reveal several noticeable local conformational variations, such as redistribution of helical twist and base pair roll between the central GpC steps and the adjacent G-tract segments, as well as a substantial helical twist variability in the CpA(TpG) steps combined with a large positive base pair roll. These local variations are rather different from those seen in the crystal.

Solution structure of a five-adenine bulge loop within a DNA duplex.

The three-dimensional solution structure of a DNA molecule of the sequence 5'-d(GCATCGAAAAAGCTACG)-3' paired with 5'-d(CGTAGCCGATGC)-3' containing a five-adenine bulge loop (dA(5)-bulge) between two double helical stems was determined by 2D (1)H and (31)P NMR, infrared, and Raman spectroscopy. The DNA in both stems adopt a classical B-form double helical structure with Watson-Crick base pairing and C2'-endo sugar conformation. In addition, the two dG/dC base pairs framing the dA(5)-bulge loop are formed and are stable at least up to 30 degrees C. The five adenine bases of the bulge loop are localized at intrahelical positions within the double helical stems. Stacking on the double helical stem is continued for the first four 5'-adenines in the bulge loop. The total rise (the height) of these four stacked adenines roughly equals the diameter of the double helical stem. The stacking interactions are broken between the last of these four 5'-adenines and the fifth loop adenine at the 3'-end. This 3'-adenine partially stacks on the other stem. The angle between the base planes of the two nonstacking adenines (A10 and A11) in the bulge loop reflects the kinking angle of the global DNA structure. The neighboring cytosines opposite the dA(5)-bulge (being parts of the bulge flanking base pairs) do not stack on one another. This disruption of stacking is characterized by a partial shearing of these bases, such that certain sequential NOEs for this base step are preserved. In the base step opposite the loop, an extraordinary hydrogen bond is observed between the phosphate backbone of the 5'-dC and the amino proton of the 3'-dC in about two-thirds of the conformers. This hydrogen bond probably contributes to stabilizing the global DNA structure. The dA(5)-bulge induces a local kink into the DNA molecule of about 73 degrees (+/-11 degrees ). This kinking angle and the mutual orientation of the two double helical stems agree well with results from fluorescence resonance energy transfer measurements of single- and double-bulge DNA molecules.

High base pair opening rates in tracts of GC base pairs.

Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.

Bradykinin antagonists with dehydrophenylalanine analogues at position 5.

Continuing the studies on structural requirements of bradykinin antagonists, it has been found that analogues with dehydrophenylalanine (deltaPhe) or its ring-substituted analogues (deltaPhe(X)) at position 5 act as antagonists on guinea pig pulmonary artery, and on guinea pig ileum. Because both organs are considered to be bradykinin B2 receptor tissues, the analogues with deltaPhe or deltaPhe(X) at position 5, but without any replacement at position 7, seem to represent a new structural type of B2 receptor antagonist. All the analogues investigated act as partial antagonists; they inhibit the bradykinin-induced contraction at low concentrations and act as agonists at higher concentrations. Ring substitutions by methyl groups or iodine reduce both the agonistic and antagonistic activity. Only substitution by fluorine gives a high potency. Incorporation of deltaPhe into different representative antagonists with key modifications at position 7 does not enhance the antagonist activity of the basic structures, with one exception. Only the combination of deltaPhe at position 5 with DPhe at position 7 increases the antagonistic potency on guinea pig ileum by about one order of magnitude. Radioligand binding studies indicate the importance of position 5 for the discrimination of B2 receptor subtypes. The binding affinity to the low-affinity binding site (KL) was not significantly changed by replacement of Phe by deltaPhe. In contrast, ring-methylation of deltaPhe results in clearly reduced binding to KL. The affinity to the high-affinity binding site (KH) was almost unchanged by the replacement of Phe in position 5 by deltaPhe, whereas the analogue with 2-methyl-dehydrophenylalanine completely failed to detect the KH-site. The peptides were synthesized on the Wang-resin according to the Fmoc/Bu(t) strategy using Mtr protection for the side chain of Arg. The dehydrophenylalanine analogues were prepared by a strategy involving PyBop couplings of the dipeptide unit Fmoc-Gly-deltaPhe(X)-OH to resin-bound fragments.

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.

Structure determination and analysis of helix parameters in the DNA decamer d(CATGGCCATG)2 comparison of results from NMR and crystallography.

The solution structure of the DNA decamer (CATGGCCATG)2 has been determined by NMR spectroscopy and restrained molecular dynamic and distance geometry calculations. The restrainted data set includes interproton distances and torsion angles for the deoxyribose sugar ring which were obtained by nuclear Overhauser enhancement intensities and quantitative simulation of cross-peaks from double quantum filtered correlation spectroscopy. The backbone torsion angles were constrained using experimental data from NOE cross-peaks, 1H-1H and 1H-31P-coupling constants. The NMR structure and the crystal structure of the DNA decamer deviates from the structure of the canonical form of B-DNA in a number of observable characteristics. Particularly, both structures display a specific pattern of stacking interaction in the central GGC base triplet. Furthermore, a specific local conformation of the TG/CA base-pair step is present in NMR and crystal structure, highlighting the unusually high flexibility of this DNA duplex part. The solution structure of the TG/CA base-pair step obtained by our high resolution NMR study is characterized by a positive roll angle, whereas in crystal this base-pair step tends to adopt remarkably high twist angles.

Computer analysis of phytochrome sequences and reevaluation of the phytochrome secondary structure by Fourier transform infrared spectroscopy.

A repertoire of various methods of computer sequence analysis was applied to phytochromes in order to gain new insights into their structure and function. A statistical analysis of 23 complete phytochrome sequences revealed regions of non-random amino acid composition, which are supposed to be of particular structural or functional importance. All phytochromes other than phyD and phyE from Arabidopsis have at least one such region at the N-terminus between residues 2 and 35. A sequence similarity search of current databases indicated striking homologies between all phytochromes and a hypothetical 84.2-kDa protein from the cyanobacterium Synechocystis. Furthermore, scanning the phytochrome sequences for the occurrence of patterns defined in the PROSITE database detected the signature of the WD repeats of the beta-transducin family within the functionally important 623-779 region (sequence numbering of phyA from Avena) in a number of phytochromes. A multiple sequence alignment performed with 23 complete phytochrome sequences is made available via the IMB Jena World-Wide Web server (http://www.imb-jena.de/PHYTO.html). It can be used as a working tool for future theoretical and experimental studies. Based on the multiple alignment striking sequence differences between phytochromes A and B were detected directly at the N-terminal end, where all phytochromes B have an additional stretch of 15-42 amino acids. There is also a variety of positions with totally conserved but different amino acids in phytochromes A and B. Most of these changes are found in the sequence segment 150-200. It is, therefore, suggested that this region might be of importance in determining the photosensory specificity of the two phytochromes. The secondary structure prediction based on the multiple alignment resulted in a small but significant beta-sheet content. This finding is confirmed by a reevaluation of the secondary structure using FTIR spectroscopy.

Hairpin-dimer equilibrium of a parallel-stranded DNA hairpin: formation of a four-stranded complex.

The 24mer deoxyoligonucleotide 3'-d(T)10-5'-5'-d(C)4- d(A)10-3'(psC4) with an uncommon 5'-p-5'phosphodiester linkage was designed to enable the formation of a hairpin structure with unusual parallel-stranded stem. As reference hairpin structure with an antiparallel-stranded stem, the 24mer 5'-d(T)10-d(C)4-d(A)10-3'(apsC4) was chosen. The behaviour of these oligonucleotides at different temperatures, DNA and salt concentrations was characterised by a combination of UV melting, CD, CD melting, infrared and Raman spectroscopy, infrared melting and analytical ultracentrifugation. The parallel-stranded hairpin structure was found to be formed by psC4 only under conditions of low DNA concentration and low salt concentration. Increase of the NaCl concentration beyond the physiological level or high DNA concentration supports the formation of intermolecular multi-stranded structures. The experimental data are in agreement with a four-stranded complex formed by two molecules of psC4. The base pairing model of this asymmetric four-stranded complex is based on the pyrimidine motif of a triple helix with two bifurcated hydrogen bonds at the O4 of the thymine each directed towards one of the amino protons of both adenines. In contrast, the reference oligonucleotide apsC4 forms only an antiparallel-stranded hairpin under all experimental conditions.

FTIR spectroscopic studies of oligonucleotides that model a triple-helical domain in self-splicing group I introns.

Fourier Transform infrared (FTIR) spectroscopy was used to characterize the Mg2+ dependent association of a 23-mer mixed ribo-deoxyribonucleotide (23-mer RNA) and a 7-mer oligoribonucleotide (7-mer RNA) that models the triple-helical domain of a self-splicing group I intron [Sarkar et al. (1996) Biochemistry 35, 4678-4688]. To elucidate the effect of deoxyribose substitution in the entire backbone, as well as at specific positions, in the assembly of the triple-helical domain, parallel studies were carried out on the association of pure deoxyribonucleotides having base sequences corresponding to the oligoribonucleotides and also between 23-mer RNA and two 7-mer RNA variants. In the variants, either the ribose attached to G451 or the ribose attached to U453 was changed to deoxyribose. FTIR-monitored thermal denaturation of the two 23-mer hairpins shows two distinct melting regions in 1 M NaCl, in case of the RNA hairpin but not for the 23-mer DNA. Triple-helix association between the two strands (7-mer and 23-mer) studied by FTIR show that only when both strands are RNA, association takes place with the formation of the P6 helix. Our results also show that the interactions between the two RNA strands involve some participation of the riboses, which could also involve the 2'-OH groups of the RNA backbone. The assembly of the triple-helical domain is not possible with a deoxyribose backbone and is completely perturbed even when only one ribose at either G451 or U453 position is substituted by deoxyribose.

Effects of ryegrass on biodegradation of hydrocarbons in soil.

The effects of growing ryegrass (Lolium perenne L.) on the biodegradation of hydrocarbons was studied in laboratory scale soil columns. Degradation of hydrocarbons as well as bacterial numbers, soil respiration rates and soil dehydrogenase activities were determined. In the rhizosphere soil system, aliphatic hydrocarbons disappeared faster than in unvegetated columns. Abiotic loss by evaporation was of minor significance. Elimination of pollutants was accompanied by an increase in microbial numbers and activities. The microbial plate counts and soil respiration rates were substantially higher in the rhizosphere than in the bulk soil. The results indicate that biodegradation of hydrocarbons in the rhizosphere is stimulated by plant roots.

A correlation between thermal stability and structural features of staphylokinase and selected mutants: a Fourier-transform infrared study.

Variants of recombinant staphylokinase (Sak) were investigated by Fourier-transform infrared spectroscopy: Sak (wild type), Sak-M26A, Sak-M26L, and Sak-G34S/R36G/R43H (Sak-B). Estimation of the secondary structure and hydrogen-deuterium exchange experiments revealed the existence of fast-exchanging and strongly solvent-exposed fractions of the helical structures in the two samples Sak and Sak-M26L. These two samples are also thermally less stable with unfolding transition temperatures of 43.7 degrees C (Sak) and 43.5 degrees C (Sak-M26L), respectively. On contrast, Sak-M26A and Sak-G34S/R36G/R43H have a slower hydrogen-deuterium exchange, have a smaller solvent-exposed portion of the helical part, and are more resistant against thermal unfolding; the transition temperatures are 51.7 degrees C and 59.3 degrees C, respectively. The secondary structure analysis was performed by two different approaches, by curve-fitting after band narrowing and by pattern recognition (factor analysis) based upon reference spectra of proteins with known crystal structure. Within the limits of the used methods, we are unable to detect significant differences in the secondary structure of the four variants of Sak. According to the results of the factor analysis, the portions of secondary structure elements were obtained to 16-20% alpha-helix, 28-30% beta-sheet, 23-27% turns, 28-30% irregular (random) and other structure. The sharp differences in the specific plasminogen-activating capacity (Sak, Sak-G34S/R36G/R43H and Sak-M26L are fully active, but Sak-M26A does not form a stable complex with plasminogen) are not reflected in the structural features revealed by the infrared spectra of this study.