Biomedical subjects

Ulrike Alexiev

Publications and source records attributed to Ulrike Alexiev.

9 recordsLinked to original sources

PubMed2006

Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin.

Understanding the coupling between conformational changes in the intramembrane domain and at the membrane-exposed surface of the bovine photoreceptor rhodopsin, a prototypical G protein-coupled receptor (GPCR), is crucial for the elucidation of molecular mechanisms in GPCR activation. Here, we have combined Fourier transform infrared (FTIR) and fluorescence spectroscopy to address the coupling between conformational changes in the intramembrane region around the retinal and the environment of helix 8, a putative cytosolic surface switch region in class-I GPCRs. Using FTIR/fluorescence cross-correlation we show specifically that surface alterations monitored by emission changes of fluorescein bound to Cys316 in helix 8 of rhodopsin are highly correlated with (i) H-bonding to Asp83 proximal of the retinal Schiff base but not to Glu122 close to the beta-ionone and (ii) with a metarhodopsin II (MII)-specific 1643 cm(-1) IR absorption change, indicative of a partial loss of secondary structure in helix 8 upon MII formation. These correlations are disrupted by limited C-terminal proteolysis but are maintained upon binding of a transducin alpha-subunit (G(talpha))-derived peptide, which stabilizes the MII state. Our results suggest that additional C-terminal cytosolic loop contacts monitored by an amide II absorption at 1557 cm(-1) play a functionally crucial role in keeping helix 8 in the position in which its environment is strongly coupled to the retinal-binding site near the Schiff base. In the intramembrane region, this coupling is mediated by the H-bonding network that connects Asp83 to the NPxxY(x)F motif preceding helix 8.

Aspartic Acid↗

PubMed2005

Simulation of fluorescence anisotropy experiments: probing protein dynamics.

Time-resolved fluorescence anisotropy decay experiments on a protein-attached dye can probe local protein dynamics and steric restrictions, but are difficult to interpret at the structural level. Aiming at an atomistic description, we have carried out molecular dynamics simulations of such experiments. Our simulations describe an Alexa488 fluorescent dye maleimide derivative covalently attached via a single cysteine to the AB-loop of bacteriorhodopsin. Fluorescence anisotropy decay curves obtained from the simulations agree well with the measured ones. Three anisotropy decay components were resolved and assigned to: 1), the fast dynamics of the attached dye on the picosecond timescale; 2), the slower dynamics of the loop at the one nanosecond timescale; and 3), the overall tumbling of the molecule. For the biologically relevant 1-ns component we identified two processes from simulations, the motion of the flexible loop as well as slow conformational dynamics of the dye. These two processes are not separable by experiment alone. Furthermore, analysis of the correlation between the dye and the protein motion revealed which part and which motion of the protein is actually probed by the experiment. Finally, our simulations allowed us to test the usual and inevitable assumption underlying these types of spectroscopic measurements that the attached dye probe does not severely perturb the protein dynamics. For the case at hand, by comparison with a simulation of the dye-free protein, the perturbation was quantified and found to be small.

Anisotropy↗

PubMed2005

G-protein-coupled receptor domain overexpression in Halobacterium salinarum: long-range transmembrane interactions in heptahelical membrane proteins.

The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.

Amino Acid Sequence↗

PubMed2005

Picosecond-pulse-induced two-photon fluorescence enhancement in biological material by application of grating waveguide structures.

We report enhancement of two-photon fluorescence (TPF) excitation in fluorescent dyes and fluorescently labeled biomolecules by exploiting the optical properties of double grating waveguide structures (DGWSs). Picosecond laser pulses generate a large evanescent field based on the guided mode phenomenon in the resonant DGWSs, which induces strong TPF signals from fluorescent dyes at the waveguide surface. By recording enhanced TPF signals of Rhodamine B and Lucifer Yellow under resonance conditions, a detection sensitivity of concentrations of approximately one dye molecule per 0.1 microm2 was achieved. For the first time to our knowledge, enhanced TPF signals of a Lucifer Yellow-labeled biomolecule (human self-peptide) in an aqueous environment are demonstrated. These results strongly encourage the use of DGWSs as enhancement platforms in modern biophysics and biotechnology for investigations of biological membranes and cells.

Equipment Design↗

PubMed2005

Thermodynamic and structural equivalence of two HLA-B27 subtypes complexed with a self-peptide.

The F pocket of major histocompatibility complex (in humans HLA) class I molecules accommodates the C terminus of the bound peptide. Residues forming this pocket exhibit considerable polymorphism, and a single difference (Asp116 in HLA-B*2705 and His116 in HLA-B*2709 heavy chains) confers differential association of these two HLA-B27 subtypes to the autoimmune disease ankylosing spondylitis. As peptide presentation by HLA molecules is of central importance for immune responses, we performed thermodynamic (circular dichroism, differential scanning calorimetry, fluorescence polarization) and X-ray crystallographic analyses of both HLA-B27 subtypes complexed with the epidermal growth factor response factor 1-derived self-peptide TIS (RRLPIFSRL) to understand the impact of the Asp116His exchange on peptide display. This peptide is known to be presented in vivo by both subtypes, and as expected for a self-peptide, TIS-reactive cytotoxic T lymphocytes are absent in the respective individuals. The thermodynamic analyses reveal that both HLA-B27:TIS complexes exhibit comparable, relatively high thermostability (Tm approximately 60 degrees C) and undergo multi-step unfolding reactions, with dissociation of the peptide in the first step. As shown by X-ray crystallography, only subtle structural differences between the subtypes were observed regarding the architecture of their F pockets, including the presence of distinct networks of water molecules. However, no consistent structural differences were found between the peptide presentation modes. In contrast to other peptides displayed by the two HLA-subtypes which show either structural or dynamical differences in their peptide presentation modes, the TIS-complexed HLA-B*2705 and HLA-B*2709 subtypes are an example for thermodynamic and structural equivalence, in agreement with functional data.

Binding Sites↗

PubMed2004

Differential peptide dynamics is linked to major histocompatibility complex polymorphism.

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.

Entropy↗

PubMed2002

Light-induced changes in the structure and accessibility of the cytoplasmic loops of rhodopsin in the activated MII state.

Bovine rhodopsin was specifically labeled on the cytoplasmic surface at cysteine 140 (the first residue of the loop connecting helices III and IV) or at cysteine 316 (in the loop connecting helix VII and the palmitoylation sites) with the fluorescent labels fluorescein and Texas Red. These loops are involved in activation and signal transduction. The time-resolved fluorescence depolarization was measured in the dark state and in the M(II) state, with labeled samples consisting of rhodopsin-octylglucoside micelles or rod outer segment (ROS) membranes. In this way the diffusional dynamics of the flexible loops of rhodopsin were measured for the first time directly on the nanosecond time scale. Control experiments showed that the large number of weak excitation pulses required in these single photon counting experiments leads to <5% bleaching of the sample. Rhodopsin was trapped in the activated M(II) state for the duration of the fluorescence experiments ( approximately 20 min) after illumination at pH 6 and 5 degrees C. For both types of samples and at both labeled positions the dynamics of the label and loop motion as monitored by the time constants of the depolarization were not significantly different in the two states of the receptor. The end-anisotropy increased, however, from 0.09 in the dark to 0.16 in the M(II) state for ROS samples labeled at C140. The corresponding numbers for the C316 position are 0.06 and 0.12. Light-induced activation in M(II) is thus associated with a large increase in the loop steric hindrance due to a changed loop domain structure on the cytoplasmic surface. These results are supported by fluorescence quenching experiments with I(-), which indicate a significant decrease in the collisional quenching constant k(q) and in accessibility in the M(II) state at both positions. The rotational correlation time of the rhodopsin micelles increased from 48 ns in the dark state to 60 ns in M(II). This increase is caused by a change in volume and/or shape and is consistent with a structural change. These results demonstrate that time-resolved fluorescence depolarization is a powerful tool to study the changes in conformation and dynamics of the cytoplasmic loops that accompany the activation of rhodopsin and other G-protein coupled receptors.

Animals↗

PubMed2002

Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

BACKGROUND: Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR. RESULTS: A pR purification procedure is described that utilizes Phenylsepharose and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent. CONCLUSIONS: The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

Cysteine↗

PubMedDate not supplied

Picosecond multidimensional fluorescence spectroscopy: a tool to measure real-time protein dynamics during function.

Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico- to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is light-activated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPC-module. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.

Amino Acid Substitution↗