Two-state expansion and collapse of a polypeptide

The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers is continuous and multi-phasic. We have used a laser temperature-jump with fluorescence spectroscopy to measure the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.     

Two-state expansion and collapse of a polypeptide

The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers and random heteropolymers is continuous and multi-phasic. A new rapid-mixing flow technique has been used to resolve the late stages of polypeptide collapse, at time scales >/=45 microseconds. We have used a laser temperature-jump with fluorescence spectroscopy to resolve the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.     

Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe.

The use of the long-lived fluorescence probe coronene (mean value of tau(FL) approximately 200 ns) is described for investigating submicrosecond lipid dynamics in DPPC model bilayer systems occurring below the lipid phase transition. Time-resolved fluorescence emission anisotropy decay profiles, measures as a function of increasing temperature toward the lipid-phase transition temperature (T(C)), for coronene-labeled DPPC small unilamellar vesicles (SUVs), are best described in most cases by three rotational decay components (phi(i = 3)). We have interpreted these data using two dynamic lipid bilayer models. In the first, a compartmental model, the long correlation time (phi(N)) is assigned to immobilized coronene molecules located in "gel-like" or highly ordered lipid phases (S-->1) of the bilayer, whereas a second fast rotational time (phi(F) approximately 2-5 ns) is associated with probes residing in more "fluid-like" regions (with corresponding lower ordering, S-->0). Interests here have focused on the origins of an intermediate correlation time (50-100 ns), the associated amplitude (beta(G)) of which increases with increasing temperature. Such behavior suggests a changing rotational environment surrounding the coronene molecules, arising from fluidization of gel lipid. The observed effective correlation time (phi(EFF)) thus reflects a discrete gel-fluid lipid exchange rate (k(FG)). A refinement of the compartmental model invokes a distribution of gel-fluid exchange rates (d(S,T)) corresponding to a distribution of lipid order parameters and is based on an adapted Landau expression for describing "gated" packing fluctuations. A total of seven parameters (five thermodynamic quantities, defined by the free energy versus temperature expansion; one gating parameter (gamma) defining a cooperative "melting" requirement; one limiting diffusion rate (or frequency factor: d(infinity))) suffice to predict complete anisotropy decay curves measured for coronene at several temperatures below the phospholipid T(C). The thermodynamic quantities are associated with the particular lipid of interest (in this case DPPC) and have been determined previously from ultrasound studies, thus representing fixed constants. Hence resolved variables are r(O), temperature-dependent gate parameters (gamma), and limiting diffusion rates (d(infinity)). This alternative distribution model is attractive because it provides a general probe-independent expression for distributed lipid fluctuation-induced probe rotational rates occurring within bilayer membranes below the phospholipid phase transition on the submicrosecond time scale.     

Single-pair fluorescence resonance energy transfer of nucleosomes in free diffusion: optimizing stability and resolution of subpopulations

We applied fluorescence detection methods on the single-molecule level to study structural variations and dynamic processes occurring within nucleosomes. Four fluorescent nucleosome constructs were made by attaching donor and acceptor fluorophores to different positions of two nucleosome positioning sequences and reconstituting nucleosomes by salt dialysis. The photochemical and biochemical stability of nucleosomes under single-molecule conditions was optimized by adding inert protein and free radical capturing additives, allowing us to define the best experimental conditions for single-molecule spectroscopy on highly diluted solutions of nucleosome complexes. We could demonstrate for the first time the resolution of conformational subpopulations of nucleosomes by single-pair fluorescence resonance energy transfer in a freely diffusing system and could show the effect of thermally induced nucleosome repositioning.     

Fast and slow gating relaxations in the muscle chloride channel CLC-1

Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from -160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to -140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, tau(f), was steeper than that of the slow one, tau(s) (apparent activation valences were z(f) approximately -0. 79 and z(s) approximately -0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (tau(f) approximately 16 micros, tau(s) approximately 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695-706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero "offsets" at negative voltages, indicating that the two "gates" never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pH(int), and [Cl](ext) dependence, and the dominant myotonia inducing mutation, I290M. Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.     

Distinct constrictive processes, separated in time and space, divide caulobacter inner and outer membranes

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.     

Ultrafast transient absorption studies on Photosystem I reaction centers from Chlamydomonas reinhardtii. 1. A new interpretation of the energy trapping and early electron transfer steps in Photosystem I

The energy transfer and charge separation kinetics in core Photosystem I (PSI) particles of Chlamydomonas reinhardtii has been studied using ultrafast transient absorption in the femtosecond-to-nanosecond time range. Although the energy transfer processes in the antenna are found to be generally in good agreement with previous interpretations, we present evidence that the interpretation of the energy trapping and electron transfer processes in terms of both kinetics and mechanisms has to be revised substantially as compared to current interpretations in the literature. We resolved for the first time i), the transient difference spectrum for the excited reaction center state, and ii), the formation and decay of the primary radical pair and its intermediate spectrum directly from measurements on open PSI reaction centers. It is shown that the dominant energy trapping lifetime due to charge separation is only 6-9 ps, i.e., by a factor of 3 shorter than assumed so far. The spectrum of the first radical pair shows the expected strong bleaching band at 680 nm which decays again in the next electron transfer step. We show furthermore that the early electron transfer processes up to approximately 100 ps are more complex than assumed so far. Several possibilities are discussed for the intermediate redox states and their sequence which involve oxidation of P700 in the first electron transfer step, as assumed so far, or only in the second electron transfer step, which would represent a fundamental change from the presently assumed mechanism. To explain the data we favor the inclusion of an additional redox state in the electron transfer scheme. Thus we distinguish three different redox intermediates on the timescale up to 100 ps. At this level no final conclusion as to the exact mechanism and the nature of the intermediates can be drawn, however. From comparison of our data with fluorescence kinetics in the literature we also propose a reversible first charge separation step which has been excluded so far for open PSI reaction centers. For the first time an ultrafast 150-fs equilibration process, occurring among exciton states in the reaction center proper, upon direct excitation of the reaction center at 700 nm, has been resolved. Taken together the data call for a fundamental revision of the present understanding of the energy trapping and early electron transfer kinetics in the PSI reaction center. Due to the fact that it shows the fastest trapping time observed so far of any intact PSI particle, the PSI core of C. reinhardtii seems to be best suited to further characterize the electron transfer steps and mechanisms in the reaction center of PSI.     

Reversible folding of rhodanese. Presence of intermediate(s) at equilibrium

For the first time completely reversible unfolding was achieved for guanidinium chloride-denatured rhodanese using a systematically defined protocol. These conditions included beta-mercaptoethanol, lauryl maltoside, and sodium thiosulfate. All components were required to get more than the previous best reactivation with lauryl maltoside of 17% (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15681). Non-coincidental transition curves were obtained by monitoring different parameters including: (i) variation in the activity, (ii) shifts of the fluorescence wavelength maximum, and (iii) variation in ellipticity at 220 nm. The transition followed by the fluorescence wavelength maximum was asymmetric and resolvable into two separate transitions. A thermodynamic analysis was used to define the energetics of the two processes. Studies with the fluorescent "apolar" probe 1,8ANS are consistent with the appearance of organized hydrophobic surfaces following the first transition. Near UV CD measurements indicated that the first transition is associated with a loss of dyssymmetry around at least some of the tryptophans. Thus, the unfolding of rhodanese is complex, and there are detectable intermediate(s) during the process. These results suggest that reversible unfolding occurs in two discrete stages: 1) loss of tertiary interactions and activity, with retention of secondary structure, and 2) loss of secondary structure. The available x-ray structure suggests that the first transition can be associated with changes in the domain interactions, which may modulate the effectiveness of helix dipoles in lowering the pKa of the active site sulfhydryl.     

Analysis of picosecond laser induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach.

A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law.     

Different modes of growth cone collapse in NG 108-15 cells

In the fundamental process of neuronal path-finding, a growth cone at the tip of every neurite detects and follows multiple guidance cues regulating outgrowth and initiating directional changes. While the main focus of research lies on the cytoskeletal dynamics underlying growth cone advancement, we investigated collapse and retraction mechanisms in NG108-15 growth cones transiently transfected with mCherry-LifeAct and pCS2+/EMTB-3XGFP for filamentous actin and microtubules, respectively. Using fluorescence time lapse microscopy we could identify two distinct modes of growth cone collapse leading either to neurite retraction or to a controlled halt of neurite extension. In the latter case, lateral movement and folding of actin bundles (filopodia) confine microtubule extension and limit microtubule-based expansion processes without the necessity of a constantly engaged actin turnover machinery. We term this previously unreported second type fold collapse and suggest that it marks an intermediate-term mode of growth regulation closing the gap between full retraction and small scale fluctuations.     

Collapse temperature of freeze-dried Lactobacillus bulgaricus suspensions and protective media

Optimization of the freeze-drying process needs to characterize the physical state of frozen and dried products. A protocol to measure the collapse temperature of complex biological media such as concentrated lactic acid bacteria using freeze-drying microscopy was first elaborated. Afterward, aqueous solutions of one or several components as well as concentrated lactic acid bacterial suspensions were analyzed in order to study how the structure of these materials is degraded during freeze-drying. A similar behavior toward collapse was observed for all aqueous solutions, which was characterized by two temperatures: the "microcollapse" temperature (T(microc), beginning of a local loss of structure) and the "collapse" temperature (T(c), beginning of an overall loss of structure). For aqueous solutions, these two temperatures were close, differing by less than 3 degrees C. Nevertheless, when lactic acid bacteria were added to aqueous solutions, the collapse temperatures increased. Moreover, the interval between microcollapse and collapse temperatures became larger. Lactic acid bacterial cells gave a kind of "robustness" to the freeze-dried product. Finally, comparing glass transition, measured by differential scanning calorimetry (DSC) and collapse temperature for aqueous solutions with noncrystallizable solutes, showed that these values belonged to the same temperature range (differing by less than 5 degrees C). As suggested in the literature, the glass transition temperature can thus be used as a first approximation of the collapse temperature of these media. However, for lactic acid bacterial suspensions, because the difference between collapse and glass transition temperatures was about 10 degrees C, this approximation was not justified. An elegant physical appearance of the dried cakes and an acceptable acidification activity recovery were obtained, when applying operating conditions during freeze-drying in vials that allowed the product temperature to be maintained during primary drying at a level lower than the collapse temperature of lactic acid bacterial suspensions. Consequently, the collapse temperature T(c) was proposed as the maximal product temperature preserving the structure from macroscopic collapse and an acceptable biological activity of cells.     

Programmed cell death of plant tracheary elements differentiating in vitro

Summary We used various microscopic and labeling techniques to examine events occurring during the programmed cell death (PCD) of plant tracheary elements (TEs) developing in vitro. TEs differentiating in vitro synthesize a secondary cell wall which is complex in composition and pattern at approximately 72 h after hormone manipulation. The timing of PCD events was established relative to this developmental marker. Cytoplasmic streaming continues throughout secondary wall synthesis, which takes 6 h to complete in a typical cell. Vital dye staining and ultrastructural analysis show that the vacuole and plasma membrane are intact during secondary cell wall synthesis, but the cytoplasm becomes less dense in appearance, most likely through the action of confined hydrolysis by small vacuoles which are seen throughout the cell at this time. The final, preeminent step of TE PCD is a rapid collapse of the vacuole occurring after completion of secondary cell wall synthesis. Vacuole collapse is an irreversible commitment to death which results in the immediate cessation of cytoplasmic streaming and leads to the complete degradation of cellular contents, which is probably accomplished by release of hydrolytic enzymes sequestered in the vacuole. This event represents a novel form of PCD. The degradation of nuclear DNA is detectable by TUNEL, an in situ labeling method, and appears to occur near or after vacuole collapse. Our observations indicate that the process of cellular degradation that produces the hollow TE cell corpse is an active and cell-autonomous process which is distinguishable morphologically and kinetically from necrosis. Although TE PCD does not resemble apoptosis morphologically, we describe the production of spherical protoplast fragments by cultured cells that resemble apoptotic bodies but which are not involved in TE PCD. We also present evidence that, unlike the hypersensitive response (HR), TE PCD does not involve an oxidative burst. While this evidence does not exclude a role for reactive oxygen intermediates in TE PCD, it does suggest TE PCD is mechanistically distinct from cell death during the HR.Abbreviations BA 6-benzylamino-purine - DAPI 4,6-diamidino-2-phenylindole diacetate - DCF 2,7-dichlorofluorescein diacetate - DPI diphenyleneiodonium - FDA fluorescein diacetate - HR hypersensitive response - NAA -naphthalene-acetic acid - PCD programmed cell death - ROI reactive oxygen intermediate - TE tracheary element - TUNEL TdT-mediated dUTP nick end labeling     

Folding mechanism of Pseudomonas aeruginosa cytochrome c551: role of electrostatic interactions on the hydrophobic collapse and transition state properties.

We report on the folding kinetics of the small 82 residue cytochrome c551from Pseudomonas aeruginosa. The presence of two Trp residues (Trp56 and Trp77) allows the monitoring of fluorescence quenching on refolding in two different regions of the protein. A single His residue (the iron-coordinating His16) permits the study of refolding in the absence of miscoordination events. After identification of the kinetic traps (Pro isomerization and aggregation of denatured protein), overall refolding kinetics is described by two processes: (i) a burstphase collapse (faster than milliseconds) which we show to be a global event leading to a state whose compactness depends on the overall net charge; at the isoeletric pH (4.7), it is maximally compact, while above and below it is more expanded; and (ii) an exponential phase (in the millisecond time range) leading to the native protein via a transition state(s) possibly involving the formation of a specific salt bridge between Lys10 and Glu70, at the contact between the N and C-terminal helices. Comparison with the widely studied horse cytochrome c allows the discussion of similarities and differences in the folding of two proteins which have the same "fold" despite a very low degree of sequence homology (<30 %).     

Linearized kinetic model of<Emphasis Type="Italic"> Listeria monocytogenes</Emphasis> biofilm growth

In this study the dynamics of biofilm formation on aluminum has been investigated. The process of cell growth has been observed using fluorescence microscopy. It has been confirmed that the process of biofilm formation can be represented as a sum of two separate processes: cell adhesion and colony proliferation. The derived set of equations describes kinetics of surface population growth and characteristic times for adsorption and combined growth processes, including characteristic time for the nutrient supply depletion. All equations contain variables based on the fundamental characteristics of bacterial population and can be easily determined from the experimental data or estimated theoretically. The developed theoretical model allows obtaining realistic values for population growth time and characteristic time for nutrient limitation occurrence during the biofilm development. Resulting equations qualitatively describe the biofilm formation process, and allow predicting microbial kinetics in the batch reactor system and determining critical values of the process parameters.     

A ligand-induced conformational change in apolipoprotein(a) enhances covalent Lp(a) formation

Lipoprotein(a) (Lp(a)) assembly proceeds via a two-step mechanism in which initial non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede disulfide bond formation. In this study, we used analytical ultracentrifugation, differential scanning calorimetry, and intrinsic fluorescence to demonstrate that in the presence of the lysine analog epsilon-aminocaproic acid, apo(a) undergoes a substantial conformational change from a "closed" to an "open" structure that is characterized by an increase in the hydrodynamic radius (approximately 10%), an alteration in domain stability, as well as a decrease in tryptophan fluorescence. Although epsilon-aminocaproic acid is a well characterized inhibitor of the non-covalent interaction between apo(a) and low density lipoprotein, we report the novel observation that this ligand at low concentrations (100 microm-1 mm) significantly enhances covalent Lp(a) assembly by altering the conformation of apo(a). We developed a model for the kinetics of Lp(a) assembly that incorporates the conformational change as a determinant of the efficiency of the process; this model quantitatively explains our experimental observations. Interestingly, an analogous conformational change has been previously described for plasminogen resulting in an increase in the hydrodynamic radius, an increase in tryptophan fluorescence, and an acceleration of the rate of plasminogen activation. Although the functions of apo(a) and plasminogen have diverged considerably, elements of structural and conformational homology have been retained leading to similar regulation of two unrelated biological processes.     

Real-time fluorescence measurement of cell-free endosome fusion: regulation by second messengers.

A quantitative real-time assay of cell-free endosomal vesicle fusion was developed and applied to study fusion mechanisms in endosomes from baby hamster kidney (BHK-21) cells. The assay is based on an irreversible approximately 10-fold increase in BODIPY-avidin fluorescence on binding of biotinylated conjugates. BODIPY-avidin and biotin-dextran were internalized for 10 min at 37 degrees C into separate populations of BHK-21 cells, and endosome fractions were prepared. Postnuclear supernatant fractions underwent ATP- and temperature-dependent fusion, as measured in a sensitive custom-built microfluorimeter by the continuous increase in BODIPY-avidin fluorescence. Fusion processes of efficiency > 2.5% could be detected with 200-ms time resolution in sample volumes of 50 microL containing endosomes derived from approximately 4 x 10(4) cells. The fusion time course consisted of a distinct lag phase (up to 10 min) in which little fusion occurred, followed by an approximately exponential rise (t 1/2 10-30 min; fusion efficiency approximately 15%). The lag phase was reduced by preincubation of separate endosome fractions with ATP at 37 degrees C and by coincubation of endosomes at 22 degrees C before the assay, suggesting a rate-limiting step involving binding of a soluble protein to the endosome membrane. Endosome fusion was strongly inhibited by GTP gamma S, N-ethylmaleimide, and AIF4-. Endosome fusion was not affected by phorbol myristate acetate but was significantly inhibited by cAMP and bovine brain calmodulin. The results establish a sensitive real-time fluorescence assay to quantify the kinetics and extent of endosome fusion in a cell-free system and demonstrate regulation of early endosome fusion by cytosolic second messengers.     

Photopolymerization as an innovative detection technique for low-density microarrays

One limitation that accounts in part for the scarcity of commercially available diagnostic microarrays is the expense associated with fluorescence detection. Here we present a colorimetric method based on photopolymerization as an "on-chip" signal amplification technique. Proof of principle experiments are detailed and followed by the use of a simple influenza microarray to demonstrate the technique for the first time with clinical samples. The advantages of this new technique include rapid (<5 min) signal amplification ( approximately 105) in ambient conditions for both DNA and protein microarrays, low reagent cost (<$1 per assay), visual or inexpensive detection, and signal preservation for at least two years under ambient conditions.     

Osmotic control of bilayer fusion

We have used photography and capacitance measurement to monitor the steps in the interaction and eventual fusion of optically black lipid bilayers (BLMs), hydrostatically bulged to approximately hemispherical shape and pushed together mechanically. A necessary first step is drainage of aqueous solution from between the bilayers to allow close contact of the bilayers. The drainage can be controlled by varying the osmotic difference across the bilayers. If the differences are such as to remove water from between the bilayers, fusion occurs after a time that depends on the net osmotic difference and the area of contact. If there is an osmotic flow of water into the space between the bilayers, fusion never occurs. In the fusion process, a single central bilayer forms from the original apposed pair of bilayers. The central bilayer may later burst to allow mixing of the two volumes originally bounded by the separate bilayer; the topological equivalent of exocytosis.     

Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism

Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision. The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping.