Mutation And Covalent Modification Increases The Porosity Of The Aquifex Aeolicus Lumazine Synthase Capsid:Redox-Triggered Encapsulation And Release Of Peptides And Oligonucleotides

In nature,protein cages are often used as nanocontainers to store,transport,or regulate guest molecules.For example,the cage formed by the lumazine synthase from Aquifex aeolicus(AaLS)assembles into a hollow dodecahedron composed of 60 identical subunits.Naturally,AaLS encapsulates the A.aeolicus riboflavin synthase homotrimer(Aa RS),which occupies the interior space of the capsid.This complex catalyzes the last two steps of riboflavin biosynthesis,with both reactions occurring inside the cage,as the AaLS active site is located at the interior surface and the Aa RS is encapsulated within the interior space.The capsid contains 12 identical five-fold symmetric channels,which are 0.9 nm in diameter,that have been thought provide the means for the substrates and products to diffuse across the shell of the AaLS cage.AaLS has also been engineered to encapsulate different types of molecular guests.However,loading guests into the capsid in vitro typically requires that the guest be able to enter the intact capsid.Therefore,the porosity of this capsid is of interest for both understanding its natural function and for developing novel encapsulation complexes.Recently,a variant of AaLS,called AaLS-IC,was engineered to position a unique cysteine per subunit at the interior surface of the cage.AaLS-IC contains two point mutations,C37 A and E122 C.In previous work,AaLS-IC,which forms a cage assembly with the same size and shape as wild-type AaLS,was used to encapsulate a thiol-modified derivative of curcumin(Cur-SH)via a two-step thiol-disulfide exchange process.In the first step,reduced AaLS-IC is oxidized by 5,5′-dithiobis-2-nitrobenzoic acid(DTNB)to generate an activated mixed disulfide adduct between the protein and NTB(AaLS-IC-NTB).Cur-SH was covalently captured inside the cage by displacing NTB from AaLS-IC-NTB.Here,I used this thiol-disulfide exchange chemistry to examine the porosity of AaLS-IC-NTB by screening a panel of seven different thiol-containing molecules with a wide range of molecular weights.The panel includes dithiothreitol(DTT,154 Da),reduced glutathione(GSH,307 Da),two dye-labeled peptide isomers(FITCAa RS12 and FITC-Aa RS12-scr,2418 Da each),a thiol-modified deoxyoligonucleotide(as Akt-1,6296 Da),the protein Escherichia coli thioredoxin(Trx,11,700 Da),and the protein bovine serum albumin(BSA,66,400 Da).The ability of the panel members to release NTB from AaLS-IC-NTB was determined using UV-Vis spectroscopy,by measuring the resulting increase in A412 nm.Of the seven panel members,only BSA was unable to access the interior of the intact cage.DTT,GSH,FITC-Aa RS12-scr,and as Akt-1 could release all of the NTB.Both Trx and FITCAa RS12 react more slowly with the capsid interior,giving only partial release of NTB from the intact capsid after 1 hour(approximately one-third and one-half,respectively).Previously,peptides which are similar to,but slightly shorter than,FITC-Aa RS12 and FITC-Aa RS12-scr have been shown to be unable to enter the intact wild-type AaLS cage.Thus,AaLS-IC-NTB displays a surprisingly high porosity.The influence of the denaturant guanidine hydrochloride on capsid porosity is also investigated here.A previous study with wild-type AaLS showed that treatment with 3 M Gdn·HCl increased its porosity,apparently via reversible partial dissociation of pentameric subunits.I repeated the screen of the thiol panel in 3 M Gdn·HCl.As before,DTT,GSH,and FITC-Aa RS12-scr could fully displace NTB from AaLS-ICNTB.Under this condition,FITC-Aa RS12,Trx,and BSA could also fully displace NTB.In contrast,the yield of NTB released by as Akt-1 decreased by about half under this condition,relative to the absence of Gdn·HCl.Nonetheless,the addition of Gdn·HCl allows larger molecules to access the capsid interior.The encapsulation of FITC-AaRS12,FITC-AaRS12-scr,as Akt-1,and BSA by AaLS-IC are characterized in greater detail.These potential guests were encapsulated by reaction with AaLS-IC-NTB in 3 M Gdn·HCl,followed by removal of the denaturant to regenerate the intact cage and trap the guest inside.After the cages were re-isolated by size-exclusion chromatography,encapsulation yields of the peptides and oligonucleotide were measured by UV-Vis spectroscopy.For the peptides and oligonucleotide,the encapsulation yields could be tuned somewhat,based on the input ratio of thiol to AaLS-IC-NTB mixed disulfide,but the number of encapsulated guests per cage were typically lower than the number of NTB molecules that had been displaced.This discrepancy may be due to inefficient re-incorporation of the guestmodified subunits into the cage upon denaturant removal.Attempts to encapsulate BSA proved unsuccessful.Even at a ratio of one BSA per cage,no BSA could be detected by SDS-PAGE analysis of the reassembled cages,although it could be detected in a later-eluting peak from the size-exclusion column that contained lowerorder assemblies of AaLS-IC.It is likely that modification of AaLS-IC with BSA sterically hinders cage incorporation of the modified subunits into the capsid assembly.After cage loading,the disulfide bond between AaLS-IC and guest can be reduced.For the FITC-Aa RS12,FITC-Aa RS12-scr,and as Akt-1 guests,disulfide bond reduction under non-denaturing conditions triggers release of a majority of the guest molecules.Thus,these molecules can also diffuse out of the intact capsid.The ability to encapsulate peptides and oligonucleotides into AaLS-IC and release these cargoes in a controlled fashion via a redox trigger may be useful for targeted delivery of drugs or imaging agents into cells.This work thus advances our understanding of the AaLS capsid and extends the range of guest molecules that can be encapsulated by this container,which may increase its utility for applications in drug delivery or other areas of bionanotechnology.