Distinct Intermediate States Research Articles

Characterization of Two Distinct β2-Microglobulin Unfolding Intermediates that May Lead to Amyloid Fibrils of Different Morphology

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.

Bacteriorhodopsin photocycle kinetics analyzed by the maximum entropy method

A maximum entropy method (MEM) was developed for the study of the bacteriorhodopsin photocycle kinetics. The method can be applied directly to experimental kinetic absorption data without any assumption for the number of the intermediate states taking part in the photocycle. Though this method does not give a specific kinetics, its result is very useful for selection between possible photocycle kinetics. Using simulated data, it is shown that MEM gives correct results for the number of the intermediate states and the amplitude distributions around the characteristic lifetimes. Analyzing experimental absorption data at five different wavelengths, MEM gives seven or eight characteristic lifetimes, which means that at least so many distinct intermediate states exist during the photocycle. Many possible photocycle kinetic models were studied and compared with the MEM result. The best agreement was found with a branching photocycle model of eight intermediate states (K, L, M1, M2, M3, M4, N, O). The branching occurs at the L intermediate state (M1 and M2 being in one branch and M3 and M4 in the other branch), but at high pH it occurs already at the K state.

Bacteriorhodopsin photocycle kinetics analyzed by the maximum entropy method

A maximum entropy method (MEM) was developed for the study of the bacteriorhodopsin photocycle kinetics. The method can be applied directly to experimental kinetic absorption data without any assumption for the number of the intermediate states taking part in the photocycle. Though this method does not give a specific kinetics, its result is very useful for selection between possible photocycle kinetics. Using simulated data, it is shown that MEM gives correct results for the number of the intermediate states and the amplitude distributions around the characteristic lifetimes. Analyzing experimental absorption data at five different wavelengths, MEM gives seven or eight characteristic lifetimes, which means that at least so many distinct intermediate states exist during the photocycle. Many possible photocycle kinetic models were studied and compared with the MEM result. The best agreement was found with a branching photocycle model of eight intermediate states ( K, L, M 1, M 2, M 3, M 4, N, O). The branching occurs at the L intermediate state ( M 1 and M 2 being in one branch and M 3 and M 4 in the other branch), but at high pH it occurs already at the K state.

X-ray crystallography of rhodopsin

Structural studies on retinal proteins have advanced significantly in recent years. Among the proteins whose structure has been solved by X-ray crystallography, rhodopsin is the only one from eukaryotic organisms having visual function. The structural model of rhodopsin also represents the first atomic template for a much larger superfamily of G protein-coupled receptors. Its natural abundance in vertebrate retinal rod cells and a novel single-step purification method made it possible to obtain three-dimensional crystals for X-ray diffraction study. The ground state structure has been refined so far to 2.6 Å resolution, by which the details of the hepta-helical transmembrane region are resolved including functional internal water molecules. Possible structural change upon photo-excitation is likely to involve interaction changes between retinal chromophore and the surrounding residues. Further studies with microspectroscopy and X-ray diffraction with improved crystals that diffract to 2 Å resolution will reveal a series of conformational changes represented by distinct intermediate states.

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions.

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.

박테리아로돕신의 광순환 특성을 이용한 광학적 논리회로 구현

본 연구에서는 bacteriorhodopsin(bR)이 첨가된 고분자 박막을 이용하여 광 논리회로 특성을 구현하였다. bR은 분광학적으로 구분되는 몇 단계의 중간단계로 구성된 복잡한 참순환 과정을 수반하고 있다. 우리는 bR의 광순환 과정중 B상태와 M상태의 흡수도 변화를 고려하여 He-Ne 레이저(632.8 nm)와 He-Cd 레이저(413 nm) 이용하여 광학적 논리회로를 구현하였다. 또한 B상태와 K상태간의 흡수 스펙트럼을 고려하여 He-Ne 레이저(632.8 nm)와 Nd-YAG 레이저의 제2고조파인 532 nm를 이용하여 고속 AND logic gate를 구현하였다. We realized optical logic gates using a bacteriorhodopsin (bR) doped polymer film. The bR undergoes a complex photocycle characterized by several spectroscopically distinct intermediate states. We realized optical logic gates using a He-Ne laser (632.8 nm) and a He-Cd laser (413 nm) that consider B-state and absorption change of M-state in the photocycle of bR. Also, we realized high speed AND logic gate using He-Ne laser (632.8 nm) and the second harmonics at 532 nm from a pulsed Nd-YAG laser that considering absorption spectrum between B-state and K-state.

Frozen motion of gliding bacteria outlines inherent features of the motility apparatus.

High-resolution data of actively gliding wild-type bacteria of four different species and of four different gliding mutants of Myxococcus xanthus were obtained from scanning electron micrographs. By shock freezing and freeze drying, motility-associated surface patterns could be fixed and consequently distinct intermediate states of motion could be observed for the first time. It is shown that these topographic patterns are immediately lost when gliding motility is stopped by blocking the respiratory chain with potassium cyanide or sodium azide. From the surface topography, the mode of action of the gliding apparatus of all four bacterial species examined can be described as a twisted circularly closed 'band'. During gliding, groups of nodes of the supertwisted apparatus show evidence of travelling like waves along the trichomes. However, the spacing between the nodes is not constant but varies within a certain range. This indicates that they are flexibly modulated as a consequence of the gliding state of the individual trichome.

Equilibrium and kinetic folding of pigeon lysozyme.

In the present study, the search for a possible intermediate state in pigeon lysozyme is addressed by equilibrium and kinetic experiments using static and stopped-flow fluorescence and circular dichroism spectroscopies. In equilibrium conditions at pH 7.5, pigeon lysozyme shows no populated intermediate state in temperature- and GdnHCl-induced unfolding experiments. In the unfolding process at low pH, however, a distinct intermediate state with molten globule characteristics is observed. Ca2+ binding to the protein is found to stabilize the native state. The early folding intermediate observed in kinetic experiments corresponds to the equilibrium intermediate in that an important amount of secondary structure has already been established. Full accomplishment of native tertiary contacts is achieved in a fast exponential process with a rate constant (0.23-135 s-1) that is strongly dependent on refolding conditions. Binding experiments with the fluorescent inhibitor MeU-diNAG support these conclusions. The folding rate is not influenced by Ca2+ binding. Analysis of the refolding and unfolding kinetics determined as a function of denaturant concentration leads to a Gibbs energy profile with a rate-determining transition state between the N- and I-states. Comparison with previous results on the folding of hen egg white lysozyme emphasizes the crucial role of Trp 62 in stabilizing non-native interactions. The replacement of this residue by Tyr in pigeon lysozyme contributes to the formation of native tertiary contacts.

Photo-excitation by a half-carotenoid: symbiosis between retinal and the visual protein opsin

Abstract

Immortal, non-tumourigenic mouse mammary outgrowths express high levels of cyclin B1 and activation of cyclin B1/cdc2 kinase.

Neoplastic transformation of mouse mammary epithelial cells is the result of several identifiable phenotypic changes which presumably require sequential genetic alterations. In our model system, mammary cells progress from a mortal state (virgin duct) to several morphologically distinct intermediate states. The intermediate states are distinct cell populations that are phenotypically identified as immortal, non-tumourigenic (i.e. EL11), weakly tumourigenic ductal/alveolar hyperplasia (i.e. EL12) and moderately tumourigenic alveolar hyperplasiaa (i.e. TM12) to invasive tumours (i.e. EL12T/TM12T). We have studied the changes in total cyclin A and B1 levels, cyclin A and B1 complexed to cdc2, cyclin B1cdc2 kinase activity and cyclin D proteins in EL11 and EL12 immortalized outgrowth lines. Results revealed increased levels in total cyclin B1 (> 5-fold), cyclin B1/cdc2 (3-4-fold) and cyclin B1/cdc2 kinase activity (2-3.5-fold) in EL11 and EL12 phenotypes when compared to control mammary gland (virgin). No changes in the levels of total cyclin A or cycln A associated to cdc2 were observed. Cyclin D1, D2 and D3 protein levels were low in the EL11 immortal ductal outgrowth. Exposure to hormones via a pituitary isograft stimulated the synthesis of cyclin D1 and D2 but not D3 associated to cdk4 as well as total cdk4 proteins. Bromodeoxyuridine (BrdUrd) labelling indices showed marked increases in immortal ductal outgrowths (EL11 and EL12) when compared to virgin, suggesting that epithelial cells are cycling in these cell populations. Even in the presence of hormone stimulation, EL11 outgrowths were not tumourigenic, suggesting that other events are necessary to drive the cells to a tumourigenic phenotype. The results suggest that increased levels of cyclin B1 and cyclin B1-cdc2 kinase activities are early events and may be an important marker for the immortalized phenotype.

Conformational changes of E. coli RNA polymerase during transcription initiation

Escherichia coli RNA polymerase-promoter complex undergoes a multistep process to initiate transcription. We have employed fluorescence spectroscopic approaches to detect the conformational states of the enzyme during this multistep process. A fluorescence assay based on the measurement of fluorescence of free and promoter-bound enzyme as a function of temperature within the range of 4 to 37°C showed that, starting with initial ‘closed complex’, there are conformationally two distinct intermediate states of the polymerase till it attains the final form required for transcription initiation. The equilibrium from closed complex (RP c) to open complex (RP o) consists of at least the following two intermediate complexes: RP c ⇄ 4−13°C RP i1 ⇄ 13−19°C RP i2 ⇄ 19−24°C RP o Higher order structure of RNAP in each of these complexes was probed by means of measurement of accessibilities of the tryptophan fluorophores to the acrylamide. In the next part of the study, TbGTP, a fluorescent substrate, has been used to probe the state of active site in the enzyme for the complexes RP c, RP i1, RP i2 and RP o, respectively. From the comparison of changes in the parameters such as, fluorescence polarization anisotropy of TbGTP and its accessibility to the neutral quencher, acrylamide, in free and promoter-bound enzyme, we have further substantiated the first part of our results. Together these results suggest that formations of RP c and RP i1 do not involve radical conformational changes in the enzyme, while the enzyme undergoes major change in conformation in the steps RP i1 → RP i2 and RP i2 → RP o. The strong tryptophan promoter cloned in plasmid pDR720 was chosen as a model promoter in these studies.

Production, Inhibitory Activity, Folding and Conformational Analysis of an N‐terminal and an Internal Deletion Variant of Chicken Cystatin

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin.

Different changes in spontaneous field potential oscillations precede epileptiform bursting in hippocampal slices perfused with penicillin or reduced magnesium

Power spectra were used to analyse spontaneous field potentials (SFPs) recorded in the CA3 distal apical dendritic region of guinea pig hippocampal slices perfused with either penicillin or reduced Mg 2+. High concentrations of penicillin (2000 IU/ml) progressively converted the low amplitudem irregular oscillations observed in control medium to higher amplitude, low frequency, rhythmic oscillations at apporximately 2–3 Hz just prior to the onset of spontaneous, synchronized bursting. Low concentrations (50–300 IU/ml) increased the power of frequencies below 10 Hz and suppressed higher in a dose-dependent fashion. Although Mg 2+-free medium also increased the magnitude of the SFPs prior to the onset of synchronous bursting, the changes were smaller than with penicillin and the frequency distribution was completely different. Low concentrations of Mg 2+ (0.0–0.5 mM) increased the power across all frequencies, however, the maximal effect was on frequencies between 5 and 25 Hz. The transition from normal to epileptiform activity may proceed through at least 2 distinct intermediate states. When recurrent inhibition is blocked (penicillin), synchronous synaptic activity precedes the onset of bursting, whereas non-specific increases in excitability and activation of NMDA receptors (reduced Mg 2+) produce an asynchronous transition state.

NMR studies identify four intermediate states of ATPase and the ion transport cycle of sarcoplasmic reticulum Ca 2+-ATPase

Water proton nuclear relaxation measurements are used to detect and characterize four distinct intermediate states for Gd 3+ bound to Ca 2+ sites of sarcoplasmic reticulum Ca 2+-ATPase in complexes with ATP analogues. In the absence of nucleotides, Gd 3+ binds to two occluded Ca 2+ transport sites on Ca 2+-ATPase which have a low accessibility to solvent water. In the presence of the nonhydrolyzable ATP analogue, Co(NH 3) 4AMPPCP, a new state for bound Gd 3+ (still occluded and with fewer waters of hydration) is observed. In the presence of Co(NH 3) 4ATP or ATP, two additional states for bound Gd 3+ are detected in the NMR studies. The first of these probably represents an intermediate state for bound Gd 3+ during ATP hydrolysis. The latter is the most occluded Gd 3+ site yet observed in these studies and is probably analogous to the highly occluded E 1-P state observed with CrATP [(1987) Biochim. Biophys. Acta 898, 313–322].