Allergenic Asteraceae in air particulate matter: quantitative DNA analysis of mugwort and ragweed.

Mugwort (Artemisia vulgaris) and ragweed (Ambrosia artemisiifolia) are highly allergenic Asteraceae. They often cause pollen allergies in late summer and fall. While mugwort is native to Europe, ragweed reached Europe as a neophyte from North America about 150 years ago and continued spreading ever since. To understand possible relationships between the spread of ragweed, its abundance in air, and to judge possible health risks for the public, we quantified ragweed DNA in inhalable fine as well as in coarse air particulate matter. Mugwort was chosen for comparison, as it is closely related to ragweed and grows in similar, though mainly not identical, habitats but is native to Germany. The DNA quantification was performed on atmospheric aerosol samples collected over a period of 5 years in central Europe. The DNA concentrations were highest during the characteristic pollination periods but varied greatly between different years. In the inhalable fine particle fraction, ragweed exceeds the mugwort DNA concentration fivefold, while the coarse particle fraction, bearing intact pollen grains, contains more mugwort than ragweed DNA. The higher allergenic potential of ragweed might be linked to the humidity or long-range transport-induced bursting of ragweed pollen into smaller allergenic particles, which may reach the lower airways and cause more intense allergic reactions. Airborne ragweed DNA was detected also outside the local pollination periods, which can be explained by atmospheric long-range transport. Back-trajectory analyses indicate that the air masses containing ragweed DNA during winter had originated in regions with milder climate and large ragweed populations (Southern France, Carpathian Basin).

Pulsed EPR determination of water accessibility to spin-labeled amino acid residues in LHCIIb.

Membrane proteins reside in a structured environment in which some of their residues are accessible to water, some are in contact with alkyl chains of lipid molecules, and some are buried in the protein. Water accessibility of residues may change during folding or function-related structural dynamics. Several techniques based on the combination of pulsed electron paramagnetic resonance (EPR) with site-directed spin labeling can be used to quantify such water accessibility. Accessibility parameters for different residues in major plant light-harvesting complex IIb are determined by electron spin echo envelope modulation spectroscopy in the presence of deuterated water, deuterium contrast in transversal relaxation rates, analysis of longitudinal relaxation rates, and line shape analysis of electron-spin-echo-detected EPR spectra as well as by the conventional techniques of measuring the maximum hyperfine splitting and progressive saturation in continuous-wave EPR. Systematic comparison of these parameters allows for a more detailed characterization of the environment of the spin-labeled residues. These techniques are applicable independently of protein size and require approximately 10-20 nmol of singly spin-labeled protein per sample. For a residue close to the N-terminus, in a domain unresolved in the existing x-ray structures of light-harvesting complex IIb, all methods indicate high water accessibility.

Thermally activated superradiance and intersystem crossing in the water-soluble chlorophyll binding protein.

The crystal structure of the class IIb water-soluble chlorophyll binding protein (WSCP) from Lepidium virginicum is used to model linear absorption and circular dichroism spectra as well as excited state decay times of class IIa WSCP from cauliflower reconstituted with chlorophyll (Chl) a and Chl b. The close agreement between theory and experiment suggests that both types of WSCP share a common Chl binding motif, where the opening angle between pigment planes in class IIa WSCP should not differ by more than 10 degrees from that in class IIb. The experimentally observed (Schmitt et al. J. Phys. Chem. B 2008, 112, 13951) decrease in excited state lifetime of Chl a homodimers with increasing temperature is fully explained by thermally activated superradiance via the upper exciton state of the dimer. Whereas a temperature-independent intersystem crossing (ISC) rate is inferred for WSCP containing Chl a homodimers, that of WSCP with Chl b homodimers is found to increase above 100 K. Our quantum chemical/electrostatic calculations suggest that a thermally activated ISC via an excited triplet state T4 is responsible for the latter temperature dependence.

Effects of egg size, parental origin and feeding conditions on growth of larval and juvenile cod Gadus morhua.

An experimental study was performed to disentangle parental and environmental effects on the growth of Atlantic cod Gadus morhua larvae and juveniles. Eggs were collected during the spawning season from spawning pairs (families) kept separately in specially designed spawning compartments. Newly hatched larvae were released simultaneously into two mesocosms of 2,500 and 4,400 m(3). Larval growth was monitored by sampling over a 10 week period, after which juveniles were transferred to on-growing tanks, where they were tagged and kept for up to 2 years. Maternal origin was determined by individual microsatellite genotyping of the larvae (n = 3949, 24 families) and juveniles (n = 600). The results showed significant positive correlations between egg size and larval size during the whole mesocosm period. Correlations, however, weakened with time and were no longer significant at the first tank-rearing sampling at an age of 9 months. Significant family-specific differences in growth were observed. The coefficient of variation (c.v.) was calculated in order to examine variation in standard length of larvae during the mesocosm period. Inter-family c.v. was on average 69% of intra-family c.v. Differences in zooplankton densities between the two mesocosms were reflected in larval growth, condition factor and c.v. Low food abundance appeared to reduce c.v. and favour growth of larvae that showed relatively slow growth at high food abundance. It is suggested that genetically determined variation in growth potential is maintained by environmental variability.

Excited state dynamics in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower investigated by transient fluorescence spectroscopy.

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.

Pigment-pigment and pigment-protein interactions in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower.

Plants contain water-soluble chlorophyll-binding proteins (WSCPs) that function neither as antennas nor as components of light-induced electron transfer of photosynthesis but are likely constituents of regulatory protective pathways in particular under stress conditions. This study presents results on the spectroscopic properties of recombinant WSCP from cauliflower reconstituted with chlorophyll b (Chl b) alone or with mixtures of Chl a and Chl b. Two types of experiments were performed: (a) measurements of stationary absorption spectra at 77 and 298 K and CD spectra at 298 K and (b) monitoring of laser flash-induced transient absorption changes with a resolution of 200 fs in the time domain of up to 100 ps. On the basis of a theoretical analysis outlined by Renger et al. (J. Phys. Chem. B 2007, 111, 10487) the data obtained in part (a) are interpreted within a model where tetrameric WSCP binds predominantly two Chl molecules in the form of an excitonically coupled "open sandwich" dimer with a tilt angle of about 30 degrees between the chlorin planes. The time-resolved measurements on Chl a/Chl b heterodimers are described by two exponential kinetics with time constants of 400 fs and 7 ps. These kinetics are assumed to reflect a heterogeneous population of WSCPs with Chl dimers either in excitonic coupled "open sandwich" or weakly coupled geometric arrays. The 400 fs component is assigned to excited-state relaxations from the upper to the lower excitonic level of the strongly coupled "open sandwich" dimer, while the 7-8 ps component probably indicates excitation energy transfer from 1Chl b* to Chl a in a dimer array with weak coupling due to significantly longer mutual distances between the chlorin rings.

Refinement of a structural model of a pigment-protein complex by accurate optical line shape theory and experiments.

Time-local and time-nonlocal theories are used in combination with optical spectroscopy to characterize the water-soluble chlorophyll binding protein complex (WSCP) from cauliflower. The recombinant cauliflower WSCP complexes reconstituted with either chlorophyll b (Chl b) or Chl a/Chl b mixtures are characterized by absorption spectroscopy at 77 and 298 K and circular dichroism at 298 K. On the basis of the analysis of these spectra and spectra reported for recombinant WSCP reconstituted with Chl a only (Hughes, J. L.; Razeghifard, R.; Logue, M.; Oakley, A.; Wydrzynski, T.; Krausz, E. J. Am. Chem. Soc. U.S.A. 2006, 128, 3649), the "open-sandwich" model proposed for the structure of the pigment dimer is refined. Our calculations show that, for a reasonable description of the data, a reduction of the angle between pigment planes from 60 degrees of the original model to about 30 degrees is required when exciton relaxation-induced lifetime broadening is included in the analysis of optical spectra. The temperature dependence of the absorption spectrum is found to provide a unique test for the two non-Markovian theories of optical spectra. Based on our data and the 1.7 K spectra of Hughes et al. (2006), the time-local partial ordering prescription theory is shown to describe the experimental results over the whole temperature range between 1.7 K and room temperature, whereas the alternative time-nonlocal chronological ordering prescription theory fails at high temperatures. Modified-Redfield theory predicts sub-100 fs exciton relaxation times for the homodimers and a 450 fs time constant in the heterodimers. Whereas the simpler Redfield theory gives a similar time constant for the homodimers, the one for the heterodimers deviates strongly in the two theories. The difference is explained by multivibrational quanta transitions in the protein which are neglected in Redfield theory.

Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation.

The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines.

Forssman antigenicity of chemically synthesized globopentaose.

The Forssman antigenicity of a chemically synthesized globopentaose was studied. Globopentaose at 40 ng showed strong inhibitory activity for the formation of a precipitin line between globopentaosylceramide (Forssman glycolipid) and anti-Forssman rabbit antiserum, while much more pentasaccharide (7 and 100 micrograms, respectively) was required to inhibit a 50% quantitative precipitin reaction and a hemolysis reaction. An immune complex of the 3H-labeled globopentaose with anti-Forssman antibody was hardly formed. Thus, the chemically synthesized globopentaose possesses the same antigenic specificity as globopentaosylceramide but it is difficult to achieve a stable complex with Forssman antibody.

Pre-steady-state kinetics of ribosomal translocation.

The two partial reactions of elongation factor G dependent translocation, the release of deacylated tRNA from the P site and the displacement of peptidyl tRNA from the A to the P site, have been studied with the stopped-flow technique. The experiments were performed with poly(U)-programmed ribosomes from Escherichia coli carrying deacylated tRNAPhe in the P site and N-AcPhe-tRNAPhe in the A site in the presence of GTP. The kinetics of the reaction were followed by monitoring either the intensity or the polarization of the fluorescence of both wybutine and proflavine located in the anticodon loop or of proflavine located in the D loop of yeast tRNAPhe or N-AcPhe-tRNAPhe. Both displacement and release fluorescence changes could be described by three exponentials, exhibiting apparent first-order rate-constants (20 degrees C) of 2 to 5 s-1 (15 s-1, 35 degrees C), 0.1 to 0.3 s-1, and 0.01 to 0.02 s-1, measured with a saturating concentration of elongation factor G (1 microM). The activation energy for the fast process of both reactions was found to be 70 kJ/mol (17 kcal/mol), while the intermediate process exhibits an activation energy of 30 kJ/mol (7 kcal/mol). The fast step is assigned to the displacement of the N-AcPhe-tRNAPhe from the A to the P site, and to the release of the tRNAPhe from the P site. The reactions take place simultaneously to form an intermediate post-translocation complex. The latter, in the intermediate step, rearranges to form a post-translocation complex carrying the deacylated tRNAPhe in an exit site and N-AcPhe-tRNAPhe in the P site, both in their equilibrium states. In parallel, or subsequently, the deacylated tRNAPhe spontaneously dissociates from the ribosome, thus completing the translocation process. The slow process has not been assigned.

Topological arrangement of two transfer RNAs on the ribosome. Fluorescence energy transfer measurements between A and P site-bound tRNAphe.

The relative arrangement of two tRNAPhe molecules bound to the A and P sites of poly(U)-programmed Escherichia coli ribosomes was determined from the spatial separation of various parts of the two molecules. Intermolecular distances were calculated from the fluorescence energy transfer between fluorophores in the anticodon and D loops of yeast tRNAPhe. The energy donors were the natural fluorescent base wybutine in the anticodon loop or proflavine in both anticodon (position 37) and D loops (positions 16 and 17). The corresponding energy acceptors were proflavine or ethidium, respectively, at the same positions. Four distances were measured: anticodon loop-anticodon loop, 24(+/- 4) A; anticodon loop (A site)-D loop (P site), 46(+/- 12) A: anticodon loop (P site)-D loop (A site), 38(+/- 10) A: D loop-D loop, 35(+/- 9) A. Assuming that both tRNAs adopt the conformation present in the crystal and that the CCA ends are close to each other, the results are consistent with the two anticodons being bound to contiguous codons and suggest an asymmetric arrangement in which the planes of the two L-shaped molecules enclose an angle of 60 degrees +/- 30 degrees.

Effects of chlorophyll a, chlorophyll b, and xanthophylls on the in vitro assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb.

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio < or = 1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.

Study on the carbohydrate specificity of antibodies formed in rabbits to synthetic glycoproteins with the carbohydrate structure of asialo-glycophorin A.

Antibodies formed in rabbits to synthetic glycoproteins with the disaccharide structure beta-D-Gal-(1-3)-D-GalNAc, the carbohydrate moiety of desialylated glycophorin A, were investigated for their carbohydrate specificity by hemagglutination techniques and radioimmunoassay. The synthetic disaccharide was coupled to human serum albumin as carrier protein using different spacer groups and different configurations of the disaccharide spacer linkage. Radioimmunological inhibition experiments using a number of different derivatives of the carbohydrate group as inhibitors revealed a significant specificity of the antibodies for the oligosaccharide structure. However, the nature of the conjugation of the disaccharide to the carrier protein was found to be important for defining the specificity of the antibodies. Furthermore the arrangement of the hapten groups in the synthetic antigen had an important influence on the antigen-antibody reaction. No cross-reactions were observed between asialo-glycophorin A and the different synthetic antigens. Synthetic antigens provide a valuable tool for generating carbohydrate-specific antibodies. However, if the synthetic hapten molecule is small compared to the combining site of antibodies, the specificity of the immunological reaction is significantly influenced by the synthetic spacer arm or even the carrier protein.

The N-terminal domain of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for its acclimative proteolysis.

Variations in the amount of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for regulation of the uptake of light into photosystem II. An endogenous proteolytic system was found to be involved in the degradation of LHCII in response to elevated light intensities and the proteolysis was shown to be under tight regulation [Yang, D.-H. et al. (1998) Plant Physiol. 118, 827-834]. In this study, the substrate specificity and recognition site towards the protease were examined using reconstituted wild-type and mutant recombinant LHCII. The results show that the LHCII apoprotein and the monomeric form of the holoprotein are targeted for proteolysis while the trimeric form is not. The N-terminal domain of LHCII was found to be essential for recognition by the regulatory protease and the involvement of the N-end rule pathway is discussed.

Truncated recombinant light harvesting complex II proteins are substrates for a protein kinase associated with photosystem II core complexes.

Previous studies directed towards understanding phosphorylation of the chlorophyll alb binding proteins comprising light harvesting complex II (LHC II) have concentrated on a single phosphorylation site located close to the N-terminus of the mature proteins. Here we show that a series of recombinant pea Lhcb1 proteins, each missing an N-terminal segment including this site, are nevertheless phosphorylated by a protein kinase associated with a photosystem II core complex preparation. An Lhch1 protein missing the first 58 amino acid residues is not, however, phosphorylated. The results demonstrate that the LHC II proteins are phosphorylated at one or more sites, the implications of which are discussed.

Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light-harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b-antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl-binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77-K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long-wavelength absorption and 730-nm fluorescence emission. In Lhcb1 with only Chl b long-wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long-wavelength properties.

Ultrafast excitation dynamics of low energy pigments in reconstituted peripheral light-harvesting complexes of photosystem I.

Ultrafast dynamics of a reconstituted Lhca4 subunit from the peripheral LHCI-730 antenna of photosystem I of higher plants were probed by femtosecond absorption spectroscopy at 77 K. Intramonomeric energy transfer from chlorophyll (Chl) b to Chl a and energy equilibration between Chl a molecules observed on the subpicosecond time scale are largely similar to subpicosecond energy equilibration processes within LHCII monomers. However, a 5 ps equilibration process in Lhca4 involves unique low energy Chls in LHCI absorbing at 705 nm. These pigments localize the excitation both in the Lhca4 subunit and in LHCI-730 heterodimers. An additional 30-50 ps equilibration process involving red pigments of Lhca4 in the heterodimer, observed by transient absorption and picosecond fluorescence spectroscopy, was ascribed to intersubunit energy transfer.

Protein domains required for formation of stable monomeric Lhca1- and Lhca4-complexes.

The peripheral light-harvesting complex of Photosystem I consists of two subpopulations, LHCI-680 and LHCI-730. The latter is composed of the two apoproteins Lhca1 and Lhca4. Recently, reconstitution of monomeric LHCI using bacterially overexpressed Lhca1 or Lhca4 was achieved. In order to obtain insight into the structure requirements for formation of monomeric light-harvesting complexes, we produced a series of N- and C-terminal deletion mutants and used the overexpressed proteins for reconstitution experiments. We found the entire extrinsic N-terminal region dispensable for monomer formation in Lhca1 and Lhca4. Also at the C-terminus, both subunits revealed similarity since all amino acids up to the end of the fourth helix could be removed without abolishing monomer formation. In connection with former corresponding results for Lhcb1, the dispensability of these regions appears to be a general feature in LHC-formation. In LHCI, however, a stabilising effect can be ascribed to these regions since the yield of complexes was decreased. In the majority of the mutant LHCI versions no effect on pigment binding was detected. However, in the LHC with the most extensively N-terminally truncated mutant of Lhca4 a dramatic shift in the 77 K fluorescence emission to shorter wavelengths was observed. This suggests that chlorophylls involved in long wavelength fluorescence emission are located in the chlorophyll array located towards the stromal face of the thylakoid membrane assuming a pigment arrangement corresponding to that in LHCII and CP29.

Characterization of O-glycosylation sites in MUC2 glycopeptides by nanoelectrospray QTOF mass spectrometry.

Sequencing of eight O-glycosylated peptides by nanoESI-QTOF-MS/MS was carried out to provide a sensitive general characterization method for determination of glycosylation site(s) and of the type of the attached carbohydrate moiety in a single experiment. The glycopeptide structures were chosen to demonstrate the feasibility of this sensitive and accurate approach, where isobaric peptide structures either (i) with the same number of attachment sites in different position in the peptide backbone, and (ii) with the same number of sugar moieties distributed on different attachment sites in the peptide backbone, can be clearly distinguished. Beside the B-type carbohydrate sequence ions of high abundance, it is possible to register diagnostic b- and y-type glycosylated peptide ions of lower abundance due to high dynamic range of the QTOF analyser. The applicability of this approach for detailed analysis of highly clustered O-glycan structures as found in biological mucin samples is discussed.

Sensitivity enhancement in pulse EPR distance measurements.

Established pulse EPR approaches to the measurement of small dipole-dipole couplings between electron spins rely on constant-time echo experiments to separate relaxational contributions from dipolar time evolution. This requires a compromise between sensitivity and resolution to be made prior to the measurement, so that optimum data are only obtained if the magnitude of the dipole-dipole coupling is known beforehand to a good approximation. Moreover, the whole dipolar evolution function is measured with relatively low sensitivity. These problems are overcome by a variable-time experiment that achieves suppression of the relaxation contribution by reference deconvolution. Theoretical and experimental results show that this approach leads to significant sensitivity improvements for typical systems and experimental conditions. Further sensitivity improvements or, equivalently, an extension of the accessible distance range can be obtained by matrix deuteration or digital long-pass filtering of the time-domain data. Advantages and limitations of the new variable-time experiment are discussed by comparing it to the established analogous constant-time experiment for measurements of end-to-end distances of 5 and 7.5 nm on rod-like shape-persistent biradicals and for the measurement of a broadly distributed transmembrane distance in a doubly spin-labeled mutant of plant light harvesting complex II.