Analysis of self-assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus

Abstract

The formation of stable and functional surface layers (S-layers) via self-assembly of surface-layer proteins on the cell surface is a dynamic and complex process. S-layers facilitate a number of important biological functions, e.g., providing protection and mediating selective exchange of molecules and thereby functioning as molecular sieves. Furthermore, S-layers selectively bind several metal ions including uranium, palladium, gold, and europium, some of them with high affinity. Most current research on surface layers focuses on investigating crystalline arrays of protein subunits in Archaea and bacteria. In this work, several complementary analytical techniques and methods have been applied to examine structure-function relationships and dynamics for assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus: (1) The secondary structure of the S-layer protein was analyzed by circular dichroism spectroscopy; (2) Small-angle X-ray scattering was applied to gain insights into the three-dimensional structure in solution; (3) The interaction with bivalent cations was followed by differential scanning calorimetry; (4) The dynamics and time-dependent assembly of S-layers were followed by applying dynamic light scattering; (5) The two-dimensional structure of the paracrystalline S-layer lattice was examined by atomic force microscopy. The data obtained provide essential structural insights into the mechanism of S-layer self-assembly, particularly with respect to binding of bivalent cations, i.e., Mg and Ca. Furthermore, the results obtained highlight potential applications of S-layers in the fields of micromaterials and nanobiotechnology by providing engineered or individual symmetric thin protein layers, e.g., for protective, antimicrobial, or otherwise functionalized surfaces.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers

“…The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 is a perfect model protein to study crystallization pathways since the recrystallization process can only be initialized by the addition of a Ca 2+ containing buffer [15,16,21,22,24,26,27,28]. A detailed discussion of the specific role of Ca 2+ -ions (and other divalent cations) associated with conformational changes within a protein and a more generic role mediating interactions between proteins (and supports) may be found in references [27,28,29].…”

“…A detailed discussion of the specific role of Ca 2+ -ions (and other divalent cations) associated with conformational changes within a protein and a more generic role mediating interactions between proteins (and supports) may be found in references [27,28,29]. The reassembly is entropy-driven and a fascinating example of matrix assembly following a multistage, non-classical pathway in which the process of S-layer protein folding is directly linked with assembly into extended clusters [13].…”

“…The results of the CB–CB path, which may be considered as equivalent to the classical “one-step” approach, fit perfectly well to the existing literature (for reviews, see [1,2,35,36]). In the future, we would like to anticipate the generalizability of the two-step approach for all those S-layer proteins where the induction of crystallinity may be triggered by a specific buffer composition (e.g., the presence of Ca 2+ for SbpA [17,27] or slp-B53 [28]).…”

<p>Slp-coated liposomes for drug delivery and biomedical applications: potential and challenges</p>

“…In addition, random coils and β-turns comprise 5%-45% of the amino acids in the Slp sequence. These random coils, α-helices, and β-sheets constitute disordered regions in the structure of Slp, [38][39][40][41][42] which may play a key role in self-assembly on the phase interface. The disordered regions occupy almost half of the full length of Slp, and simultaneously, the high content of disordered regions results in the flexibility of Slp to alter its conformation for adapting to the phase interface, which has a crucial role in self-assembly on the surface of liposomes.…”

“…The disordered regions occupy almost half of the full length of Slp, and simultaneously, the high content of disordered regions results in the flexibility of Slp to alter its conformation for adapting to the phase interface, which has a crucial role in self-assembly on the surface of liposomes. 38,42 After treatments with detergents/breaking agents (eg, guanidine hydrochloride or urea) or application of chelating agents and cation substitution, Slp can be isolated from the bacterial cell wall due to the weak interactions between Slp subunits. 6,43,44 These Slp subunits can hold together and reassemble spontaneously on a two-phase interface or in suspension, forming specific crystalline arrays.…”