Abstract
The recombinant bacterial surface layer (S-layer) protein rSbpA of Lysinibacillus sphaericus CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered—after washing away the unbound S-layer protein—by the addition of a CaCl2 containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds. This two-step approach is new in basic and application driven S-layer research and, most likely, will have advantages for functionalizing surfaces (e.g., by spray-coating) with tailor-made biological sensing layers.
Highlights
Bacterial surface layer (S-layer) proteins have attracted much attention for more than four decades since their unique self-assembly properties allowed for functionalized solid supports, such as silicon wafers or gold surfaces, with monomolecular crystalline lattices exposing chemical groups and biologically active domains in ordered dense packing [1,2,3,4]
The aim of this study focused on the separation of S-layer lattice formation into two steps, namely on (i) a short incubation of the substrate with SbpA S-layer protein leading to a layer of adsorbed protein followed by removal of excess material; and (ii) transition of the adsorbed amorphous clusters into a crystalline array exhibiting square (p4) lattice symmetry by addition of crystallization buffer (CB) containing CaCl2 but no further S-layer protein (Figure 1)
quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) have demonstrated that the adsorption of S-layer proteins and their transition into the crystalline state follows a multi-stage non-classical pathway
Summary
Bacterial surface layer (S-layer) proteins have attracted much attention for more than four decades since their unique self-assembly properties allowed for functionalized solid supports, such as silicon wafers or gold surfaces, with monomolecular crystalline lattices (termed S-layers or S-layer lattices) exposing chemical groups and biologically active domains in ordered dense packing [1,2,3,4]. Moroeroevoevre, rQ, QCMCM-D-D usuu1a5sl0ulyadlplayrtaopvrpioodvienidstseas(mraoumucguhhclhbyebteetvetterertryitmi1me.5erser)seoswoluelurteitoiotnanktthehnaannwAAhFiFlMeM, ::afftootrrheeexxasaammmppelleet,,iwmwieti,thhoininntlyhtheaefiffiresrwts5tA5mFmiMni,nai,mraoaruognuedsnd 150wdoautladphoaivnetsb(ereonucgahpltyureevde.rMy o1.r5eosv)ewr,earsesthaokwenn winhFilieg,uaret t3hbeinsatmheentiemxte3, 0onmliyn,athfeewmAinFuMte itmrenadges wotuolwdahradvsembaesesnlocsaspatnudresdti.ffeMnoinrgeofovrert,haesCsBh-oTwrisnpianthFiwghuirlee,3ibn cinomthpearniseoxnt ,3a0wmayinf,rothmetmhoisneuftoerttrheend towCaBr–dCsBmapsasthlosws aonudldstpifrfoenbainbglyfohratvhee CbBee-Tnrischpaaltlhenwgihnigle,fionr coAmFMpa.riHsoonw, aevwear,y farsoimde thfroosme fotrhtehe CB–stCraBigphattfhorwwoaurdldapprporboaabclhyohfainvesibtueeAnFcMhaflolernsgtuindgyifnogr AcrFyMsta.lHgorowwetvhe,rt,haesriedeisfarlosmo tthheepsotrsasiigbihlittfyortwhaatrd appthroeaAchFMoftiipn msiitguhAt iFnMterfaocrt wstiuthdyinindgivcidryusatlaSl-glaryoewr tphr,ottheienrse, iisn aplasrotitchuelapr oaststihbeilmityomtheantt twheheAnFtMheytip migahret binintedriancgt twoiathcriynsdtiavlliidnueaplaSt-clha,yaenrdptrhoutesininsf,luinenpcaerttihceurlaersualttst.he moment when they are binding to a crystalline patch, and influence the results Based upon these findings, the starting point for AFM investigations were silicon chips incubated with rSbpA (50–200 μg/mL crystallization buffer) for 5 or 10 min, respectively. After washing away unbound S-layer protein, the crystalline lattice structure could clearly be seen by AFM when the coated substrates were further incubated in a crystallization buffer (average domain size 1–2 μm). When the additional incubation was carried out in Tris buffer (without CaCl2) or Milli-Q water, the S-layer lattice structure could not be visualized These findings were supported by the observation that the crystallization process or the transformation of the adsorbed amorphous into the crystalline state was terminated by crosslinking the S-layer protein clusters with glutardialdehyde (Figure S3). 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