Isolation,Microscopic Structure And Chemical Stabilization Of PC645 Complex Of Chroomonas Placoidea

The PC645 complex that had not been reported before was steadly isolated and almost purified on a large scale from Chroomonas placoidea T13 by using modified and optimized sucrose density gradient centrifugation based on the method described previously,which therefore demonstrate that cryptophytic phycobiliproteins do have native aggregation forms.The results of absorption,fluorescence,circular dichroism spectra and analysis of isoelectric point as well as peptide composition of PC complex etc.confirmed that PC complex showed basically the same spectral features as the PC645 in heterodimeric form,and was a kind of aggregate composed ofα₁α₂β₁β₁heterodimers.Theβ₂subunit found in the previous laboratory research work was not appeared in this PC complex.The resulting PC complex had a molecular weight of about 240 k Da,but all dissociated into heterodimers during secondary SDGC or low salt（0.05 mol/L PBS）elution conditions.Cross linker glutaraldehyde and condensation agent EDC were all failed in the stabilization of PC complex,while formaldehyde,a small molecule cross linker,was demonstrated more feasible in the crosslinking reaction of PC complexes.It was found that the crosslinking reaction did not affect the fluorescence emission properties of the PC complex,but preferentially affected the 645 nm absorption peak corresponding to the PCBβ₈₂phycobilin,then followed by the characteristic peaks at 580 and 625 nm,indicating that the PCBβ₈₂phycobilin should be located on the outside position of the PC complex.The formaldehyde stabilization conditions repeatedly groped for were initial optimized as follows:formaldehyde concentration 100.14 mg/m L,reaction time 6 h,complex sample A₆₄₅=2.5.The spectral changes of cross-linked products during 3-month of storage were measured.And it was found that the cross-linked products were not completely dissociated during SDS-PAGE,IEF,SDGC or in 0.05 mol/L low salt concentration PBS,and exhibited high tolerance to high concentrations of urea and SDS.A molecular sieve chromatography system with PE hexamer and PC645 heterodimer as molecular weight indexes was established,by which the molecular weight of the stabilized crosslinking product was calculated as 120 k Da,correspond to a cross-linking product of two heterodimers.The experimental results laid a foundation for the development of new chemically stabilized fluorescent probes of cryptophytic phycobiliprotein in the future.Based on the statistical analysis data of a large number of particles in top view,side view and oblique view in pictures obtained by negative staining TEM,it was recognized that the PC complex in band II is a monolayer annular particle with four PC645heterodimers put together,whose outer diameter is 11～16 nm（average 13.36±2.64 nm）,inner diameter of 2～5 nm（average 3.48±1.52 nm）,and thickness about 4～6 nm（average5±1 nm）.In addition,hollow annular particles had also been observed to aggregate fatherly via their ring side,suggesting that PC complexes may have higher level of assembly forms.On the premise that PCBβ₈₂phycobilins are located outside of the PC complex,we predicted the most likely postures of PC645 heterodimer and the amino acid distribution on both sides of the complex by using Py MOL software.And by the aid of M-ZDOCK protein docking software,assembly models involved four PC645 heterodimers were predicted.The 6th and 9th predicted poses with very similar conformations were screened out from the top 10 highest-scoring prediction models.And the parameters of pose 6（or 9）are:ring inner diameter of 3.32 nm（3.55 nm）,outer diameter 11.2 nm（11.35 nm）,height about 6.1 nm（6.24 nm）and the recess are 3.3 nm（3.63 nm）high.And in both of the two postures,each heterodimer combines with each other in the same posture as predicted by Py MOL,only 2 of the heterodimers twist a little.Both of them have 3 almost identical contact regions between their contacting surfaces,and most of them are loops or small spirals,and rich in small non-polar amino acids.Each of the phycobilin PCBβ₈₂is on the outside of the ring in the two postures,and the outer edge of the ring structures is smooth,which is consistent with the results of cryo-EM observation.The predicted postures well matched with the particle images in cryo-EM pictures.All above suggest that the PC complex can exists at least two stable conformations,posture 6 is denser,more compact,and may be more rational conformation,while pose 9 is a metastable structure.Accordingly,we proposed that the conformation of PC complex is flexible and not unique.These results break through the previous recognition about the existence states of cryptophytic phycobiliproteins,and will be of great significance for the study on the structure,function and evolution of photosynthetic systems in cryptophytes.