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Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy
KTH, School of Biotechnology (BIO), Biochemistry.
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2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 50, 16658-16671 p.Article in journal (Refereed) Published
Abstract [en]

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyi-sn-glycero-3-phosphati-dylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 1418514196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TILL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.

Place, publisher, year, edition, pages
2005. Vol. 44, no 50, 16658-16671 p.
Keyword [en]
electron-paramagnetic-resonance, candida-rugosa lipase, x-ray crystallography, interfacial activation, humicola-lanuginosa, continuous-wave, triacylglycerol lipase, conformational-changes, geotrichum-candidum, triglyceride lipase
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-15254DOI: 10.1021/bi051478oISI: 000234036300028Scopus ID: 2-s2.0-29244471711OAI: oai:DiVA.org:kth-15254DiVA: diva2:333295
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Lipase-Lipid Interactions Studied by Site-Directed Labeling and Biological Spectroscopy
Open this publication in new window or tab >>Lipase-Lipid Interactions Studied by Site-Directed Labeling and Biological Spectroscopy
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes the study of lipase–lipid interactions of the triglyceride lipase Thermomyces lanuginosus lipase, TLL, by use of site-directed chemical labeling and biological spectroscopy. Several single-cysteine variants of TLL were produced, and labeled with probes containing either an affinity-, a fluorescence- or a spin-label functionality. A combined reduction- and labeling protocol was elaborated for the TLL single-cysteine variants, which were found to be prematurely oxidized during production. A diagnostic dot-blot method based on biotin-avidin interactions was established for the detection of free thiols in recombinant proteins. This method has a picomole detection limit and was found accurate in free thiol quantitation.

Unilamellar phospholipid vesicles consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) or of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) were produced as stable, well-defined lipid interfaces, to which TLL is known to bind with high affinity. The binding orientation of TLL was investigated, employing eleven spin-labeled TLL single-cysteine variants with the point-mutations positioned around the protein surface. Spin-relaxation enhancement was induced by the spin-relaxation agent chromium(III) oxalate (Crox), and a novel ESR-method for measuring the interactions of nitroxide spin-labels at low-microwave amplitudes was established. This method requires only standard ESR-instrumentation and is optimal for surface-exposed spin-labels. The interactions of TLL spin-labels with Crox in the aqueous phase were studied by ESR spectroscopy, while spin-label interactions with the lipid phase were analyzed by fluorescence quenching of dansyl-labeled vesicles. In addition, ESR-data was employed in conjunction with electrostatic potential-based modeling to dock TLL on the membrane of small POPG vesicles. This afforded information on the detailed molecular orientation of TLL at the lipid–water interface.

TLL was found to bind on 50-nm–diameter POPG vesicles in an activated form with the active-site entrance and the proximal part of the lid oriented against the lipid membrane. On 100 nm POPG vesicles and 50 nm POPC vesicles, TLL was oriented with the active site facing away from the membrane, corresponding to approximately a 180-degree–rotation about TLL’s vertical axis. There was no deep penetration of TLL residues in the lipid membranes, but TLL was found to associate closer to the negatively charged POPG-surface, than that of the zwitterionic POPC. TLL was also able to bind to two vesicles simultaneously, as revealed by fluorescence resonance energy transfer (FRET) between fluorescent labeled vesicles. The experimental data obtained provided insights into the interfacial behaviour of a triglyceride lipase.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. x, 68 p.
Keyword
thermomyces lanuginous lipase, site-directed labeling, lipase-lipid interactions
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-618 (URN)91-7178-253-2 (ISBN)
Public defence
2006-02-24, Svedbergssalen, AlbaNova universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
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Note
QC 20100830Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2010-08-30Bibliographically approved

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