Hartmut Fritzsche

FTIR-spectroscopic characterization of phosphocholine-headgroup model compounds

Abstract The polar headgroups are an important molecular subregion of phospholipids since they mediate a substantial part of the interactions emerging with other constituents of biological membranes, be it inherent macromolecules (proteins) or water as forming the natural environment. FTIR spectroscopy is well proven for characterizing aspects of weak interactions, above all hydrogen bonding. We have used this method to study solid deposits of a number of selected lipid models, such as choline, acetylcholine and methylphosphocholine (MePC), and compared them with the common phospholipid dimyristoylglycerophosphocholine (DMPC), at different degrees of hydration, which were varied via relative humidity (RH). At low RHs, only MePC and DMPC take up considerable amounts of water, thus elucidating the essential role of phosphate groups in the first stages of lipid hydration. The progress of PC-headgroup hydration can be sensitively monitored by the wavenumber decrease of the band owing to antisymmetric PO 2 − stretching vibration. Concomitant variations of the spectral parameters of ν CH bands of MePC reveal that conformational changes may simultaneously occur in the headgroup. Surprisingly to us, the model compounds display qualitative differences in the appearance of their ν CH bands, which are most probably a result of substituent effects.

Die Gestalt der OH-Valenzschwingungsbande des Phenols bei Assoziation mit Carbonylverbindungen

Zusammenfassung The shape of the broad band due to the OH stretching vibration of hydrogen bond complexes of phenol with proton acceptors has been studied. The shape deviates from the normal symmetrical type for some classes of proton acceptors. An attempt has been made to explain these anomalous shapes. In the special case of hydrogen bonding between phenol and carbonyl compounds the asymmetrical band shape is interpreted by superposition of two symmetrical bands and connected with a hydrogen bond isomerism.

COMPARATIVE FTIR SPECTROSCOPIC STUDY UPON THE HYDRATION OF LECITHINS AND CEPHALINS

Abstract Fourier-transform infrared (FTIR) spectroscopy has been used to study the gradual hydration of films prepared from some ubiquitous phospholipids. The diacyl lecithins (PCs, DPPC and DOPC) and cephalins (PEs, DPPE and DOPE) are representative for compounds with saturated (palmitoyl) and unsaturated (oleoyl) hydrocarbon chains, respectively. The adsorption isotherms obtained spectroscopically reveal that lecithins take up more water than cephalins, independently of the nature of their acyl chains. Furthermore, whereas the two lecithins exhibit more or less substantial and continuous wavenumber shifts for the well-assigned infrared absorption bands arising from the vibrations of their polar parts, the cephalins show a significantly diverse pattern with characteristic differences determined by the chemical nature of their tails. DPPE is, as far as reflected by its IR-band parameters, completely invariant against hydration, i.e. no influence of the water imbibed by the film is visible. Such a finding can be interpreted in terms of a tight hydrogen-bonding network formed between the phosphate and ammonium groups of DPPE. This explanation is confirmed by the results of relevant quantum-chemical AM1 calculations another part of which is also suitable to rationalize the wavenumber downwards shifts of the PO−2 and CO stretching-vibration bands of lecithins observed upon hydration. DOPE differs from DPPE by undergoing rather dramatic hydration-induced wavenumber shifts of the IR bands due to its polar parts. Contrarily to the lecithins, however, these displacements are restricted to a very narrow range of water activities. This behaviour suggests the existence of a lyotropic phase transition ascribed to a conversion from H∥ to Pα occurring when the water content of DOPE is decreased.

Chain-length dependence of the hydration properties of saturated phosphatidylcholines as revealed by FTIR spectroscopy

Abstract Phospholipids — as biomembrane constituents — are an interesting class of molecules, and among them, phosphatidylcholines (PCs) or lecithins are most abundant in eucaryotic organisms. We have used FTIR spectroscopy to explore hydration-induced phenomena in symmetric, even-numbered, saturated diacyl PCs with varying chain length, n , of 10–22. These compounds were studied at room temperature as multilamellar films dependent on water activity which was regulated via the ambient relative humidity (RH). The PCs were compared in terms of water-uptake capacity by analyzing adsorption isotherms obtained from IR spectra. As demonstrated in the data, this parameter is governed by the phase type adopted in the lipid assemblies rather than by the sheer chain length. Short-chain lipids with n ≤14 which are shown to undergo lyotropic main transitions at room temperature and to adopt the lamellar liquid–crystalline phase can imbibe considerably more water than the other PCs maintaining a rigid phase at any RH. Chain melting is not systematically correlated with spectral features due to bands assigned to polar IR-active groups (phosphate, carbonyl).

Infrared spectroscopy and infrared linear dichroism of nucleic acids

Abstract The activities of this laboratory in the field of infrared spectroscopy of nucleic acids since 1964 are summarized. Vibrations of deoxyribonucleic acid (DNA) were assigned by inspection of spectra of DNAs differing in their base composition. The “melting” of DNA dissolved in D 2 O has been studied by infrared spectroscopy, and the base composition of DNA samples from different microorganisms has been determined both from absorbance differences of base vibrations and by measurrments of the “melting” temperature. Further studies have dealt with the structure of the copper(II) -DNA complex and the assignment of the out-of-plane NH vibration bands of the DNA bases. It was shown that the purine H-8 atoms undergo slow isotopic exchange at elevated temperature (55°C), allowing preparation of deuterium-labelled native DNA. The introduction of infrared linear dichroism enabled the B—A transition of oriented DNA films to be monitored as a function of the relative humidity. An infrared band at 1185 cm −1 was described as a new “marker” band for A-DNA. The infrared linear dichroism results were treated quantitatively to determine the phosphate group geometry of B-DNA and A-DNA, and to calculate the ligand-induced “freezing” of a fraction of the DNA in the B form. A new drug assay was presented based upon this “freezing” effect.

Untersuchungen zur Ermittlung von Assoziationskonstanten aus infrarotspektroskopischen Messungen an Alkoholen

Abstract Infrared-spectroscopic measurements on the free OH absorption band of isopropanol, t-butanol and t-pentanol using various concentrations in CCl4 and various temperatures, showed that the fraction α of the non-associated alcohol molecules was dependent on the concentration. Five different association models were used for calculating the formation constants of the single species. Within the limits of the accuracy of the measurements the values of α found as a function of concentration at various temperatures may be described by several contradictory models. Agreement between experiment and model may thus be accidental. Many errors and disagreements in the interpretation of association published in former studies, are comprehensible for this reason. In addition to the dimer absorption band near 3500 cm−1, these results support the view that with alcohols only a general association model with all species present is correct up to a definite maximum magnitude, and that special hypotheses concerning the value of the single formation constants may at best be considered as rough approximations.

FTIR SPECTROSCOPIC FEATURES OF LYOTROPICALLY INDUCED PHASE TRANSITIONS IN PHOSPHOLIPID MODEL MEMBRANES

Abstract Fourier-transform infrared (FTIR) spectroscopy was used to probe the effects of hydration on phospholipids arranged in multilayer films at room temperature. In 1-palmitoyl, 2-oleoylphosphatidylcholine (POPC) and 1,2-dioleoylphosphatidylethanolamine (DOPE), lyotropic phase transitions could be detected and assigned to chain melting and to a P α -H II transition involving non-lamellar structures, respectively. From a methodological point of view, lipid phase transitions like those are indicated in two different ways: (i) by drastic, abrupt changes of the indicative parameters, and, (ii), by hystereses found when non-equilibrium hydration/dehydration scans are run instead of performing the isopiestic experiments.

Parallel‐stranded duplex DNA containing dA · dU base pairs

DNA oligonucleotides with dA and dU residues can form duplexes with trans d(A.U) base pairing and the sugar-phosphate backbone in a parallel-stranded orientation, as previously established for oligonucleotides with d(A.T) base pairs. The properties of such parallel-stranded DNA (ps-DNA) 25-mer duplexes have been characterized by absorption (uv), CD, ir, and fluorescence spectroscopy, as well as by nuclease sensitivity. Comparisons were made with duplex molecules containing (a) dT in both strands, (b) dU in one strand and dT in the second, and (c) the same base combinations in reference antiparallel-stranded (aps) structures. Thermodynamic analysis revealed that total replacement of deoxythymine by deoxyuridine was accompanied by destabilization of the ps-helix (reduction in Tm by -13 degrees C in 2 mM MgCl2, 10 mM Na-cacodylate). The U-containing ps-helix (U1.U2) also melted 14 degrees C lower than the corresponding aps-helix under the same ionic conditions; this difference was very close to that observed between ps and aps duplexes with d(A.T) base pairs. Force field minimized structures of the various ps and aps duplexes with either d(A.T) or d(A.U) base pairs ps/aps and dT/dU combinations are presented. The energy-minimized helical parameters did not differ significantly between the DNAs containing dT and dU.

Solution Structure of the Dodecamer d-(CATGGGCC-CATG)2 is B-DNA. Experimental and Molecular Dynamics Study

Abstract 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. The results are discussed in terms of recent findings of DNA structures with mixed features of A-DNA and B-DNA.

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.