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Lipase-Lipid Interactions Studied by Site-Directed Labeling and Biological Spectroscopy
KTH, School of Biotechnology (BIO).
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 [en]
thermomyces lanuginous lipase, site-directed labeling, lipase-lipid interactions
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-618ISBN: 91-7178-253-2 (print)OAI: oai:DiVA.org:kth-618DiVA: diva2:14630
Public defence
2006-02-24, Svedbergssalen, AlbaNova universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100830Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2010-08-30Bibliographically approved
List of papers
1. Selective reduction and chemical modification of oxidized lipase cysteine mutants
Open this publication in new window or tab >>Selective reduction and chemical modification of oxidized lipase cysteine mutants
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2002 (English)In: Canadian journal of chemistry (Print), ISSN 0008-4042, E-ISSN 1480-3291, Vol. 80, no 6, 529-539 p.Article in journal (Refereed) Published
Abstract [en]

Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) Were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.

Keyword
lipase, cysteine mutant, selective reduction, chemical modification, methanethiosulfonate, humicola-lanuginosa lipase, fluorescence spectroscopy, conformational-changes, protein, stability, sulfhydryl, dynamics, enzymes, binding, thiols
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-21725 (URN)10.1139/v02-046 (DOI)000176891000003 ()
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved
2. Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy
Open this publication in new window or tab >>Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy
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2002 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 48, 14185-14196 p.Article in journal (Refereed) Published
Abstract [en]

The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry, 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using, a water-soluble spin relaxation agent. chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (< 0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.

Keyword
alpha/beta-hydrolase fold, candida-rugosa lipase, x-ray crystallography, humicola-lanuginosa, bacteriorhodopsin mutants, triacylglycerol lipase, conformational-changes, triglyceride lipase, substrate-binding, triad forms
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-22089 (URN)10.1021/bi020158r (DOI)000179517000012 ()
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved
3. Low microwave-amplitude ESR spectroscopy: Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
Open this publication in new window or tab >>Low microwave-amplitude ESR spectroscopy: Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
2004 (English)In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 60, no 2, 117-138 p.Article in journal (Refereed) Published
Abstract [en]

Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1) < 0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T-2e, is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-1252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T-2e. We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.

Keyword
electron spin resonance, microwave saturation, MTSL, crox, lipase, electron-paramagnetic-resonance, thermomyces-lanuginosa lipase, bacteriorhodopsin mutants, progressive saturation, membrane transporter, linewidth analysis, loop region, dynamics, liquids, conformation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-23667 (URN)10.1016/j.jbbm.2004.05.002 (DOI)000223463400003 ()2-s2.0-3242710304 (Scopus ID)
Note
QC 20100525 QC 20110923Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved
4. 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
Open this publication in new window or tab >>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
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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.

Keyword
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:nbn:se:kth:diva-15254 (URN)10.1021/bi051478o (DOI)000234036300028 ()2-s2.0-29244471711 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved

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