Atomistic Structure of Monomolecular Surface Layer Self-Assemblies: Toward Functionalized Nanostructures

Horejs, Christine-Maria; Gollner, Harald; Pum, Dietmar; Sleytr, Uwe B.; Peterlik, Herwig; Jungbauer, Alois; Tscheließnig, Rupert

doi:10.1021/nn1035729

Cited by 27 publications

(38 citation statements)

References 46 publications

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“…The protein form factor is the Fourier transform of the pair correlation of the crystallographic data of cutinase. The pair correlation was computed by taking into account all sites as possible sources of scattering (20)(21)(22).…”

Section: Methodsmentioning

confidence: 99%

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Ribitsch

Acero

Przylucka

et al. 2015

Appl Environ Microbiol

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159

102

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g Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k cat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center. P olyethylene terephthalate (PET) is the best-known and most widespread synthetic polyester in the world. It is used for foils and bottles as well as for fibers for the textile industry (1). Recycling of PET and modification of its properties for different applications by traditional procedures involve harsh chemical and physicochemical treatments (2, 3). Enzymatic modification, particularly by cutinases, has been recognized as a powerful alternative in the past decade (4, 5) and, besides offering new avenues for PET recycling, has the additional advantage of creating a modified PET with increased dyeing efficacy and improved binding to polyvinyl chloride without altering the polymer's bulk properties (4, 5).On the other hand, enzymatic hydrolysis of PET has the inherent disadvantage of occurring at a very low rate (5, 6). The reasons for this are not yet clearly understood. The access of the active center of the cutinase to the insoluble substrate is apparently one of the rate-limiting points, because enlarging the areas around the active sites of cutinases from Fus...

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Section: Methodsmentioning

confidence: 99%

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Ribitsch

Acero

Przylucka

et al. 2015

Appl Environ Microbiol

Self Cite

159

102

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“…S-layer proteins apparently adopt a different conformation when bound to their specific ligands. It has been found recently that S-layer proteins adopt different conformations as monomers and when part of self-assembled structures (8,74). Obviously, such a conformational change also takes place when S-layer proteins bind to SCWPs.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule Force Spectroscopy Reveals the Individual Mechanical Unfolding Pathways of a Surface Layer Protein

Horejs

Ristl

Tscheließnig

et al. 2011

Journal of Biological Chemistry

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Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra-and intermolecular mechanical stress. In Gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopybased single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level.

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“…Furthermore, they are involved in the interaction between bacteria and their hosts, as well as with the outer environment. S-layers act as molecular sieves that mediate exchange of nutrients and metabolites (Horejs et al 2011). In some pathogenic bacteria, S-layer proteins are also known to be key virulence factors (Kern and Schneewind 2010;Sabet et al 2003;Dooley et al 1988;Ishiguro et al 1981;Kokka et al 1990).…”

Section: Introductionmentioning

confidence: 99%

“…So far, only the three-dimensional (3D) structure of the surface-layer protein SbsB from Geobacillus stearothermophilus (G. stearothermophilus) PV72/p2 has been published (Baranova et al 2012) (PDB code 4aq1). However, to investigate potential applications, detailed information is required on the structure of S-layer proteins and their assembly (Horejs et al 2011). There are two main reasons for this lack of structural information: (1) The fundamental building blocks of S-layers consist of proteins with high molecular weight (MW), ranging from 40 to 200 kDa (Sleytr and Messner 1983), which usually are either glycosylated or possess other posttranslational modifications.…”

Section: Introductionmentioning

confidence: 99%

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

Drobot

Oberthüer

et al. 2016

Eur Biophys J

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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.

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Atomistic Structure of Monomolecular Surface Layer Self-Assemblies: Toward Functionalized Nanostructures

Cited by 27 publications

References 46 publications

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Single-molecule Force Spectroscopy Reveals the Individual Mechanical Unfolding Pathways of a Surface Layer Protein

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

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