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F Siebert

Publications and source records attributed to F Siebert.

At least 19 recordsLinked to original sources
PubMed2001

FTIR studies of phytochrome photoreactions reveal the C=O bands of the chromophore: consequences for its protonation states, conformation, and protein interaction.

The molecular changes of phytochrome during red --> far-red and reverse photoreactions have been monitored by static infrared difference spectroscopy using the recombinant 65 kDa N-terminal fragment assembled with a chromophore chemically modified at ring D or with a chromophore isotopically labeled with (18)O at the carbonyl group of ring A. This allows the identification of the C=O stretching vibrations of rings D and A. We exclude the formation of an iminoether in Pfr. The positions of both these modes show that the chromophore always remains protonated. The upshift of the C=O stretch of ring D in the first photoproducts is explained by a twisted methine bridge connecting rings C and D. The changes in the vibrational pattern during the red --> far-red conversion show that the backreaction is not just the reversal of the forward reaction. The infrared difference spectra of the fragment deviate very little from those of the full-length protein. The differences which are related to the lack of the C-terminal half of the protein constituting the signaling domain are possibly important for the understanding of the signaling mechanism.

Avena↗
PubMed2001

Anions stabilize a metarhodopsin II-like photoproduct with a protonated Schiff base.

In rhodopsin, the retinal chromophore is covalently bound to the apoprotein by a protonated Schiff base, which is stabilized by the negatively charged counterion Glu113, conferring upon it a pK(a) of presumably >16. Upon photoexcitation and conformational relaxation of the initial photoproducts, the Schiff base proton neutralizes the counterion, a step that is considered a prerequisite for formation of the active state of the receptor, metarhodopsin II (MII). We show that the pK(a) of the Schiff base drops below 2.5 in MII. In the presence of solute anions, however, it may be increased considerably, thereby leading to the formation of a MII photoproduct with a protonated Schiff base (PSB) absorbing at 480 nm. This PSB is not stabilized by Glu113, which is shown to be neutral, but by stoichiometric binding of an anion near the Schiff base. Protonation of the Schiff base in MII changes neither coupling to G protein, as assessed by binding to a transducin-derived peptide, nor the conformation of the protein, as judged by FTIR and UV spectroscopy. A PSB and an active state conformation are therefore compatible, as suggested previously by mutants of rhodopsin. The anion specificity of the stabilization of the PSB follows the series thiocyanate > iodide > nitrate > bromide > chloride > sulfate in order of increasing efficiency. This specificity correlates inversely with the strength of hydration of the respective anion species in solution and seems therefore to be determined mainly by its partitioning into the considerably less polar protein interior.

Animals↗
PubMed2001

Conformations of the active and inactive states of opsin.

The signaling state metarhodopsin II of the visual pigment rhodopsin decays to the apoprotein opsin and all-trans retinal, which are then regenerated to rhodopsin by the visual cycle. Opsin is known to have at neutral pH only a small residual constitutive activity toward its G protein transducin, which is thought to play a considerable role in light adaptation (bleaching desensitization). In this study we show with Fourier-transform infrared spectroscopy that after metarhodopsin II decay, opsin exists in two conformational states that are in a pH-dependent equilibrium at 30 degrees C with a pK of 4.1 in the presence of hydroxylamine scavenging the endogenous all-trans retinal. Despite the lack of the native agonist in its binding pocket, the low pH opsin conformation is very similar to that of metarhodopsin II and is likewise stabilized by peptides derived from rhodopsin's cognate G protein, transducin. The high pH form, on the other hand, has some conformational similarity to the inactive metarhodopsin I state. We therefore conclude that the opsin apoprotein displays intrinsic conformational states that are merely modulated by bound all-trans retinal.

Animals↗
PubMed2001

Salt dependence of the formation and stability of the signaling state in G protein-coupled receptors: evidence for the involvement of the Hofmeister effect.

We studied the salt dependence of both the stability and the equilibrium of the late photoproducts metarhodopsin I (MI) and II (MII) of the artificial visual pigment 9-demethyl rhodopsin (9dm-Rho). In the photoproducts of 9dm-Rho, all-trans-9-demethyl retinal acts only as a partial agonist, enabling us to study the photoproduct equilibrium of the pigment both in membranes and in detergent micelles. Chloride, bromide, and phosphate salts shift this equilibrium from the inactive MI to the active MII receptor conformation both in native membranes and even more with purified pigment in detergent micelles. In the presence of these salts, the induced MII state seems to be structurally intact, as judged by Fourier transform infrared (FTIR) and UV-vis spectroscopy. In the long term, however, we observe an increased instability of the photoproducts and a change in the decay pathways. Both MII enhancement and destabilization are particularly pronounced with the strong chaotropic salts KI and KSCN. The results fit into the framework of the Hofmeister effect and are assigned to an increased solvation of the peptide moiety of the solvent-exposed domains, their resulting partial disordering favoring MII over MI. In this picture, increased solvation also affects helix-helix interactions, thereby leading to a structural instability of the protein in the long term. The reported influences of salts on conformation and stability of this membrane protein are likely to be general and may therefore also apply to other transmembrane proteins and particularly to other G protein-coupled receptors.

Animals↗
PubMed2001

Static and time-resolved step-scan Fourier transform infrared investigations of the photoreaction of halorhodopsin from Natronobacterium pharaonis: consequences for models of the anion translocation mechanism.

The molecular changes during the photoreaction of halorhodopsin from Natronobacterium pharaonis have been monitored by low-temperature static and by time-resolved step-scan Fourier transform infrared difference spectroscopy. In the low-temperature L spectrum anions only influence a band around 1650 cm(-1), tentatively assigned to the C=N stretch of the protonated Schiff base of L. The analysis of the time-resolved spectra allows to identify the four states: K, L(1), L(2), and O. Between L(1) and L(2), only the apoprotein undergoes alterations. The O state is characterized by an all-trans chromophore and by rather large amide I spectral changes. Because in our analysis the intermediate containing O is in equilibrium with a state indistinguishable from L(2), we are unable to identify an N-like state. At very high chloride concentrations (>5 M), we observe a branching of the photocycle from L(2) directly back to the dark state, and we provide evidence for direct back-isomerization from L(2). This branching leads to the reported reduction of transport activity at such high chloride concentrations. We interpret the L(1) to L(2) transition as an accessibility change of the anion from the extracellular to the cytosolic side, and the large amide I bands in O as an indication for opening of the cytosolic channel from the Schiff base toward the cytosolic surface and/or as indication for changes of the binding constant of the release site.

Bacteriorhodopsins↗
PubMed2000

Transducin-dependent protonation of glutamic acid 134 in rhodopsin.

A highly conserved carboxylic acid residue in rhodopsin, Glu(134), modulates transducin (G(t)) interaction. It has been postulated that Glu(134) becomes protonated upon receptor activation. We studied the interaction between rhodopsin and G(t) using Fourier transform infrared (FTIR) difference spectroscopy combined with attenuated total reflection (ATR). Formation of the complex between G(t) and photoactivated rhodopsin reconstituted into phosphatidylcholine vesicles caused prominent infrared absorption increases at 1641, 1550, and 1517 cm(-)(1). The rhodopsin mutant E134Q was also studied. When measured in the presence of G(t), replacement of Glu(134) by glutamine abolished the low-frequency part of a broad absorption band at 1735 cm(-)(1) that is normally superimposed on the light-induced absorption changes of Asp(83) and Glu(122) of rhodopsin. In addition, a negative absorption band at 1400 cm(-)(1) that is evoked by interaction of native metarhodopsin II (MII) with G(t) was not observed in the difference spectrum of the E134Q mutant. Thus, Glu(134) is ionized in the dark and exhibits a symmetrical COO(-) stretching vibration at 1400 cm(-)(1). Glu(134) becomes protonated in the G(t)-MII complex and displays a C=O stretching mode near 1730 cm(-)(1). The E134Q mutation also affects absorption changes attributable to lipids, suggesting that the protonation of Glu(134) is linked to transfer of the carboxylic acid side chain from a polar to a nonpolar environment by becoming exposed to the lipid phase when G(t) binds. These results show directly that Glu(134) becomes protonated in MII upon G(t) binding and suggest that changes in receptor conformation affect lipid-protein interactions.

Animals↗
PubMed2000

The molecular origin of the inhibition of transducin activation in rhodopsin lacking the 9-methyl group of the retinal chromophore: a UV-Vis and FTIR spectroscopic study.

The formation of the active rhodopsin state metarhodopsin II (MII) is believed to be partially governed by specific steric constraints imposed onto the protein by the 9-methyl group of the retinal chromophore. We studied the properties of the synthetic pigment 9-demethyl rhodopsin (9dm-Rho), consisting of the rhodopsin apoprotein regenerated with synthetic retinal lacking the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by the modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equilibrium shift from MII to its immediate precursor, MI. The MII state of 9dm-Rho displays only a partial deprotonation of the retinal Schiff base, leading to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII formation is slowed by 2 orders of magnitude compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respective states in the native pigment, pointing to a more relaxed fit of the retinal chromophore in its binding pocket. The shifted equilibrium between MI and MII is therefore discussed in terms of an increased entropy of the 9dm-Rho MI state due to changed steric interactions.

Animals↗
PubMed2000

The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction.

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G(t)). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G(t) are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the beta(2)-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys(322) and Cys(323). We suggest that the amino terminus of the fourth loop interacts directly with G(t), particularly with Galpha(t), and with other regions of the intracellular surface of rhodopsin to support G(t) binding.

Amino Acid Sequence↗
PubMed2000

Structural investigation of the active site in bacteriorhodopsin: geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR.

Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.

Anisotropy↗
PubMed2000

Rhodopsin activation affects the environment of specific neighboring phospholipids: an FTIR spectroscopic study.

Rhodopsin is a member of a superfamily of G-protein-coupled receptors that transduce signals across membranes. We used Fourier-transform infrared (FTIR) difference spectroscopy to study the interaction between rhodopsin and lipid bilayer upon receptor activation. A difference band at 1744 cm(-1) (+)/1727 cm(-1) (-) was identified in the FTIR-difference spectrum of rhodopsin mutant D83N/E122Q in which spectral difference bands arising from the carbonyl stretching frequencies of protonated carboxylic acid groups were removed by mutation. As the band was abolished by detergent delipidation, we suggested that it arose from carbonyl groups of phospholipid fatty acid esters. Rhodopsin and the D83N/E122Q mutant were reconstituted into various (13)C-labeled 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles and probed. The 1744-cm(-1) (+)/1727 cm(-1) (-) band could be unequivocally assigned to a change in the lipid ester carbonyl stretch upon receptor activation, with roughly equal contribution from both lipid esters. The band intensity scaled with the amount of rhodopsin but not with the amount of lipid, excluding the possibility that it was due to the bulk lipid phase. We also excluded the possibility that the lipid band represents a change in the number of boundary lipids or a general alteration in the boundary lipid environment upon formation of metarhodopsin II. Instead, the data suggest that the lipid band represents the change of a specific lipid-receptor interaction that is coupled to protein conformational changes.

Amino Acid Substitution↗
PubMed2000

Vibrational spectroscopy as a tool for probing protein function.

Vibrational spectroscopy has become increasingly important as a tool for understanding the mechanisms of photosystem II, phytochrome and terminal oxidases. More general enzymatic or receptor systems have been studied, opening a new field of applications. Femtosecond infrared pump/probe studies of the important amide-I band seem to provide a basis for its molecular and structural interpretation.

Light↗
PubMed2000

The photoreactions of recombinant phytochrome from the cyanobacterium Synechocystis: a low-temperature UV-Vis and FT-IR spectroscopic study.

The interconvertible photoreactions of recombinant phytochrome from Synechocystis reconstituted with phycocyanobilin were investigated by light-induced optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be deferred below -100 degrees C for the transformation of red-absorbing form of phytochrome (Pr)-->far-red-absorbing form of phytochrome (Pfr), and no formation of an intermediate similar to the photoproduct of phytochrome A obtained at -140 degrees C (lumi-R) was observed. Two intermediates could be stabilized below -40 degrees C and between -40 and -20 degrees C, and were denoted as meta-Ra and meta-Rc, respectively. Above -20 degrees C Pfr was obtained. In the reverse reaction two intermediates could be stabilized below -60 degrees C (lumi-F) and between -60 and -40 degrees C (meta-F). The FT-IR difference spectra of the late Pr-->Pfr photoreaction show great similarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environment, whereas deviations in the spectra of meta-Ra were observed. A large band around 1700 cm-1 in the difference spectra between the intermediates and Pr which is tentatively assigned to the C19=O group of the prosthetic group indicates the Z,E isomerization around the C15=C16-methine bridge of the chromophore during the formation of meta-Ra. In the difference spectra of the parent states only small differences are observed in this region suggesting that the frequency of the carbonyl group is similar in Pr and Pfr. Since the FT-IR difference spectra between lumi-F and Pfr show great similarities to the spectra of the parent states, it is assumed that during the formation of lumi-F the chromophore largely returns into the primary Pr conformation. The FT-IR spectra recorded in a medium of 2H2O generally show a downshift of the significant bands due to the isotope effect. The appearance of a characteristic band around 935 cm-1 in all 2H2O spectra suggests an assignment to an N-2H bending vibration of the chromophore.

Cold Temperature↗
PubMed1999

Distortion of the L-->M transition in the photocycle of the bacteriorhodopsin mutant D96N: a time-resolved step-scan FTIR investigation.

The D96N mutant of bacteriorhodopsin has often been taken as a model system to study the M intermediate of the wild type photocycle due to the long life time of the corresponding intermediate of the mutant. Using time-resolved step-scan FTIR spectroscopy in combination with a sample changing wheel we investigated the photocycle of the mutant with microsecond time resolution. Already after several microseconds an intermediate similar to the MN state is observed, which contrasts with the M state of the wild type protein. At reduced hydration M and N intermediates similar to those of wild type BR can be detected. These results have a bearing on the interpretation of the photocycle of this mutant. A mechanism is suggested for the fast rise of MN which provides some insight into the molecular events involved in triggering the opening of the cytosolic channel also of the wild type protein.

Asparagine↗
PubMed1999

Time-resolved step-scan Fourier transform infrared spectroscopy reveals differences between early and late M intermediates of bacteriorhodopsin.

In this report, from time-resolved step-scan Fourier transform infrared investigations from 15 ns to 160 ms, we provide evidence for the subsequent rise of three different M states that differ in their structures. The first state rises with approximately 3 microseconds to only a small percentage. Its structure as judged from amide I/II bands differs in small but well-defined aspects from the L state. The next M state, which appears in approximately 40 microseconds, has almost all of the characteristics of the "late" M state, i.e., it differs considerably from the first one. Here, the L left arrow over right arrow M equilibrium is shifted toward M, although some percentage of L still persists. In the last M state (rise time approximately 130 microseconds), the equilibrium is shifted toward full deprotonation of the Schiff base, and only small additional structural changes take place. In addition to these results obtained for unbuffered conditions or at pH 7, experiments performed at lower and higher pH are presented. These results are discussed in terms of the molecular changes postulated to occur in the M intermediate to allow the shift of the L/M equilibrium toward M and possibly to regulate the change of the accessibility of the Schiff base necessary for effective proton pumping.

Bacteriorhodopsins↗
PubMed1999

Combined restorative proctocolectomy and pancreaticoduodenectomy for familial adenomatous polyposis.

This is a case report on FAP in a 41-year-old woman in which preoperative examination showed a giant tubulovillous adenoma of the descending duodenum surrounding the papilla of Vater, in addition to pancolon adenomatous polyposis, and isolated adenomas in the gastric antrum and corpus. As it was impossible to remove the giant villous adenoma in the duodenum endoscopically, prophylactic surgical treatment was chosen consisting of restorative proctocolectomy and additional pancreaticoduodenectomy. Flat tubulovillous adenomas in the gastric corpus were successfully removed by total snare biopsies before operation. FAP coli patients treated by prophylactic surgery are now known to be at risk of developing adenomas anywhere in the intestine and many affected patients later die from upper gastrointestinal tumors. In this single case report, the simultaneous occurrence of FAP coli and giant villous adenoma of the duodenum indicates the frequent outcome of this genetic alteration requiring lifelong surveillance. This rare case report includes a short survey of the relevant literature.

Adenomatous Polyposis Coli↗
PubMed1998

Evidence for the specific interaction of a lipid molecule with rhodopsin which is altered in the transition to the active state metarhodopsin II.

Comparing the FTIR difference spectra of the rhodopsin --> metarhodopsin II transition in membranes and in dodecylmaltoside detergent, characteristic variations are observed between 1715 and 1750 cm(-1). By repeating the measurements with the rhodopsin mutant D83N/E122Q, the spectral variation between the samples in membranes versus detergent could be assigned to a difference band at 1743(+)/1724(-) cm(-1), which does not exhibit a deuteration-induced downshift. We provide evidence that this band is probably caused by the C=O stretch of only one ester group of one lipid molecule. This group interacts with the dark state of rhodopsin, whereas in metarhodopsin II, the lipid molecule behaves as if it were in the bulk lipid phase.

Amino Acid Substitution↗
PubMed1998

Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin.

Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu122 and His211 on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu122 allowed assignments of the C=O stretch of protonated Glu122 in the dark state and in MI of Rho. Mutation of His211, but not of other histidine residues, affects these vibrational modes assigned to Glu122. In addition, replacements of His211 affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu122. These modes are influenced as well when Glu122 is replaced by Ala in mutant E122A but not when it is replaced by Gln in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu122 and His211.

Alanine↗