SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates

Deepika Dahiya; Zsuzsanna Péter-Szabó; Manjula Senanayake; Sai Venkatesh Pingali; Wellington C. Leite; James Byrnes; Garry W. Buchko; Pramod Sivan; Francisco Vilaplana; Emma R. Master; Hugh O’Neill

doi:10.1186/s13068-025-02618-5

SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates

Deepika Dahiya, Zsuzsanna Péter-Szabó, Manjula Senanayake, Sai Venkatesh Pingali, Wellington C. Leite, James Byrnes, Garry W. Buchko, Pramod Sivan, Francisco Vilaplana, Emma R. Master, Hugh O’Neill

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

Abstract

Background: Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples. Results: Whereas the SANS analysis of Spruce holocellulose revealed an increase in inter-microfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in the ordered arrangement of microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi β-barrel (DPBB) domain surrounded by three α-helices—the largest nestled against the DPBB core and the other two part of loops extending from the core. Conclusions: The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing regular order in the microfibril arrangement to increasing inter-microfibril spacing. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.

Original language	English
Article number	27
Journal	Biotechnology for Biofuels and Bioproducts
Volume	18
Issue number	1
DOIs	https://doi.org/10.1186/s13068-025-02618-5
State	Published - Dec 2025

Funding

This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 964764. The content presented in this document represents the views of the authors, and the Commission is not responsible for any use that may be made of the information it contains. This research was also performed on project award 60859 under the FICUS program ( https://dx.doi.org/ https://doi.org/10.46936/fics.proj.2023.60859/60008908 ) and used resources at the DOE Environmental Molecular Sciences Laboratory, which are DOE Office of Science User Facilities. The facility is sponsored by the Biological and Environmental Research program and operated under Contract No. DE-AC05-76RL01830 (EMSL). The scattering portion of this work further acknowledges the support of the Genomic Science Program, under Contract FWP ERKP752 and the Center for Structural Molecular Biology (CSMB) under Contract FWP ERKP291, Office of Biological and Environmental Research, U.S. Department of Energy and the High Flux Isotope Reactor supported by the Basic Energy Sciences, U.S. Department of Energy. The LiX beamline is part of the Center for BioMolecular Structure (CBMS), which is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1605010). LiX also received additional support from NIH Grant S10 OD012331. As part of NSLS-II, a national user facility at Brookhaven National Laboratory, work performed at the CBMS is supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences Program under contract number DE-SC0012704. This manuscript has been coauthored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript or allow others to do so, for United States Government purposes.

Keywords

Expansin
Lignocellulose
Loosenin
Small-angle neutron scattering
Solution NMR structure

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10.1186/s13068-025-02618-5

Cite this

Dahiya, D., Péter-Szabó, Z., Senanayake, M., Pingali, S. V., Leite, W. C., Byrnes, J., Buchko, G. W., Sivan, P., Vilaplana, F., Master, E. R., & O’Neill, H. (2025). SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates. Biotechnology for Biofuels and Bioproducts, 18(1), Article 27. https://doi.org/10.1186/s13068-025-02618-5

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title = "SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates",

abstract = "Background: Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples. Results: Whereas the SANS analysis of Spruce holocellulose revealed an increase in inter-microfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in the ordered arrangement of microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi β-barrel (DPBB) domain surrounded by three α-helices—the largest nestled against the DPBB core and the other two part of loops extending from the core. Conclusions: The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing regular order in the microfibril arrangement to increasing inter-microfibril spacing. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.",

keywords = "Expansin, Lignocellulose, Loosenin, Small-angle neutron scattering, Solution NMR structure",

author = "Deepika Dahiya and Zsuzsanna P{\'e}ter-Szab{\'o} and Manjula Senanayake and Pingali, \{Sai Venkatesh\} and Leite, \{Wellington C.\} and James Byrnes and Buchko, \{Garry W.\} and Pramod Sivan and Francisco Vilaplana and Master, \{Emma R.\} and Hugh O{\textquoteright}Neill",

note = "Publisher Copyright: {\textcopyright} The Author(s) 2025.",

year = "2025",

month = dec,

doi = "10.1186/s13068-025-02618-5",

language = "English",

volume = "18",

journal = "Biotechnology for Biofuels and Bioproducts",

issn = "2731-3654",

number = "1",

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Dahiya, D, Péter-Szabó, Z, Senanayake, M , Pingali, SV , Leite, WC, Byrnes, J, Buchko, GW, Sivan, P, Vilaplana, F, Master, ER & O’Neill, H 2025, 'SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates', Biotechnology for Biofuels and Bioproducts, vol. 18, no. 1, 27. https://doi.org/10.1186/s13068-025-02618-5

SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates. / Dahiya, Deepika; Péter-Szabó, Zsuzsanna; Senanayake, Manjula et al.
In: Biotechnology for Biofuels and Bioproducts, Vol. 18, No. 1, 27, 12.2025.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates

AU - Dahiya, Deepika

AU - Péter-Szabó, Zsuzsanna

AU - Senanayake, Manjula

AU - Pingali, Sai Venkatesh

AU - Leite, Wellington C.

AU - Byrnes, James

AU - Buchko, Garry W.

AU - Sivan, Pramod

AU - Vilaplana, Francisco

AU - Master, Emma R.

AU - O’Neill, Hugh

PY - 2025/12

Y1 - 2025/12

N2 - Background: Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples. Results: Whereas the SANS analysis of Spruce holocellulose revealed an increase in inter-microfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in the ordered arrangement of microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi β-barrel (DPBB) domain surrounded by three α-helices—the largest nestled against the DPBB core and the other two part of loops extending from the core. Conclusions: The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing regular order in the microfibril arrangement to increasing inter-microfibril spacing. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.

AB - Background: Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples. Results: Whereas the SANS analysis of Spruce holocellulose revealed an increase in inter-microfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in the ordered arrangement of microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi β-barrel (DPBB) domain surrounded by three α-helices—the largest nestled against the DPBB core and the other two part of loops extending from the core. Conclusions: The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing regular order in the microfibril arrangement to increasing inter-microfibril spacing. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.

KW - Expansin

KW - Lignocellulose

KW - Loosenin

KW - Small-angle neutron scattering

KW - Solution NMR structure

UR - https://www.scopus.com/pages/publications/85219598059

U2 - 10.1186/s13068-025-02618-5

DO - 10.1186/s13068-025-02618-5

M3 - Article

AN - SCOPUS:85219598059

SN - 2731-3654

VL - 18

JO - Biotechnology for Biofuels and Bioproducts

JF - Biotechnology for Biofuels and Bioproducts

IS - 1

M1 - 27

ER -

SANS investigation of fungal loosenins reveals substrate-dependent impacts of protein action on the inter-microfibril arrangement of cellulosic substrates

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