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Modeling environment effects on spectroscopic properties of biomarkers and catalytic mechanisms in enzymes
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry. KTH, Royal Institute of Technology.ORCID iD: 0000-0002-7624-4867
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Arguably, humans are in need of both better diagnostic tools to prevent pro- gression of diseases as well as greener catalysts for synthesis of chemicals.

Neurodegenerative diseases affecting neurons in the brain leads to demen- tias, where Alzheimer’s disease (AD) is the most prevalent. It is estimated that about 50 million people worldwide suffer from AD, a number that has more than doubled during the last 30 years. Currently, there is no cure for AD, but in order to slow the progression of symptoms it is crucial to develop biomarkers for early detection and initiation of clinical interventions.

With theoretical tools it is possible to better understand the optical prop- erties of fluorescent biomarkers, and thus contribute to steering the design of biomarkers for distinguishing different types of disease-associated proteins. Lu- minescent conjugated oligothiophenes (LCO) is a class of molecules that binds to aggregates of misfolded amyloid-β proteins, facilitating in vivo-detection of the pathological hallmarks of AD. By performing molecular dynamics (MD) simulations and subsequent response theory calculations of a LCO, it could be concluded that the differences in the spectroscopic fingerprints for the bound and free biomarker were predominantly due to conformational changes of the conjugated π-system in the molecular backbone. The introduction of differ- ent central units with donor properties yield donor-acceptor-donor electronic systems that increase the range of spectroscopic detection of LCO biomark- ers, without reducing the selectivity towards amyloid-β. It was also revealed that in order to capture more of the two-photon absorption (TPA) signal it would be optimal to design biomarkers with the dominant TPA signal at longer wavelenghts.

The second part of this work is centered around computational enzyme design, and how single point mutations can alter the flow of water in the active site. The altered flow of water likely impacts the catalysis in the active site of the enzymes. The enzymes considered in this work belongs to two different enzyme classes, and catalyse different kinds of reactions. Squalene hopene cyclase (SHC) is a monotopic membrane enzyme that catalyses the cyclization of squalene to hopene, and ω-transaminase catalyses the transfer of an amino and keto group between an amino acid and a keto acid. Enzyme variants of both SHC and ω-transaminase, where single-point mutations have been introduced, display different experimentally observed properties compared to their corresponding wild-types (WT). By performing MD simulations, the flow of water in the active sites of both enzymes could be tracked. Distinct differences in the flow of water in the WT and enzyme variants could be detected. These changes are proposed to influence the catalysis, and help to explain the experimentally observed differences in the protein variants.

Abstract [sv]

Behovet av bättre diagnostiska verktyg för att kunna detektera olika åldersrelaterade sjukdomar ökar. Samtidigt ökar behovet av mer miljövänliga sätt att syntetisera olika typer av kemikalier.

Neurodegenererande sjukdomar som påverkar hjärnan leder till olika typer av demens. Den vanligast förekommande formen är Alzheimers, som 50 miljoner människor uppskattas vara drabbade av. Detta är en dubblering av antalet sjukdomsfall som för 30 år sedan. I dagsläget finns inget botemedel mot Alzheimers, men det finns läkemedel som kan bromsa utvecklingen av symptomen. För att kunna starta behandlingen så tidigt som möjligt är det kritiskt att ha tillgång till biomarkörer för att kunna detektera de felveckade proteinerna som orsakar symptomen innan utvecklingen har gått för långt.

Med hjälp av simuleringar kan en djupare förståelse för de spektroskopiska egenskaperna hos fluoroscerande biomarkörer uppnås. De kunskaperna kan bidra till att styra designen av nya biomarkörer som är optimerade för att kunna detektera olika typer av sjukdomsassocierade proteiner. Luminiscerande konjugerade oligotiofener (LCO) är en grupp molekyler som binder till aggregat av felveckade amyloid- proteiner, och därmed möjliggör in vivo-detektion av de patologiska kännetecknen av Alzheimers. Genom molekyldynamiksimuleringar (MD) och efterföljande responsberäkningar av en LCO, kunde de spektroskopiska profilerna för inbunden och fri biomarkör undersökas. Det visade sig att det största bidraget härstammar från molekylernas konformation, och att bidrag från Coulomb-interaktioner mellan biomarkör och omgivningen är försumbara. Genom att introducera andra molekylära enheter istället för den centrala thiophenringen erhölls biomarkörer med ett bredare detektionsområde. Beräkningarna kunde också belysa problem med att den experimentellt detekterade signalen från två-foton spektroskopi till största delen ligger utanför det detekterade området, och att för att kunna öka möjligheterna för detektion bör designen av biomarkörer förskjutas mot molekyler som emitterar ljus vid längre våglängder.

Den andra delen av det här arbetet är centrerat kring hur punktmutationer i enzym påverkar flödet av vatten i den aktiva siten. Ett ändrat flöde föreslås påverka katalysen som utförs av enzymen. De enzym som är studerade tillhör olika enzymklasser, och katalyserar olika reaktioner. Squalene hopene cyclas (SHC) är ett monotopiskt membranenzym som katalyserar omvandlingen av skvalen till hopen. !-transaminas katalyserar reaktionen som överför en aminogrupp och en ketogrupp mellan en aminosyra och en ketosyra. För båda enzymer har punktmutationer introducerats, vilket lett till experimentellt observerade skillnader i egenskaper jämfört med respektive enzyms vildtyp (WT). Från MD simuleringar kunde flödet av vatten i den aktiva siten jämföras mellan WT och de muterade varianterna, och distinkta skillnader av vattenflöden i den aktiva siten kunde identifieras. Det ändrade flödet föreslås påverka enzymets katalytiska förmåga, vilket kan bidra till att förklara de experimentellt observerade skillnaderna hos varianterna.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2020. , p. 94
Series
TRITA-CBH-FOU ; 2020:6
Keywords [en]
Computational chemistry, spectroscopy, biocatalysis, water flow, biomarkers
National Category
Engineering and Technology
Research subject
Theoretical Chemistry and Biology
Identifiers
URN: urn:nbn:se:kth:diva-266480ISBN: 978-91-7873-422-1 (print)OAI: oai:DiVA.org:kth-266480DiVA, id: diva2:1385693
Public defence
2020-02-12, FB55, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 2020-01-20

Available from: 2020-01-20 Created: 2020-01-15 Last updated: 2020-01-20Bibliographically approved
List of papers
1. QM/MM Density Functional Theory Simulations of the Optical Properties Fingerprinting the Ligand-binding of Pentameric Formyl Thiophene Acetic Acid in Amyloid-β(1–42)
Open this publication in new window or tab >>QM/MM Density Functional Theory Simulations of the Optical Properties Fingerprinting the Ligand-binding of Pentameric Formyl Thiophene Acetic Acid in Amyloid-β(1–42)
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-266492 (URN)
Note

QC 20200115

Available from: 2020-01-15 Created: 2020-01-15 Last updated: 2020-01-15Bibliographically approved
2. Deciphering the electronic transitions of thiophene-based donor-acceptor-donor pentameric ligands utilized for multimodal fluorescence microscopy of protein aggregates
Open this publication in new window or tab >>Deciphering the electronic transitions of thiophene-based donor-acceptor-donor pentameric ligands utilized for multimodal fluorescence microscopy of protein aggregates
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-266493 (URN)
Note

QC 20200115

Available from: 2020-01-15 Created: 2020-01-15 Last updated: 2020-01-15Bibliographically approved
3. MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
Open this publication in new window or tab >>MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
Show others...
2017 (English)In: ACS Omega, ISSN 2470-1343, Vol. 2, no 11, p. 8495-8506Article in journal (Refereed) Published
Abstract [en]

Squalene–hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
National Category
Biocatalysis and Enzyme Technology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-234939 (URN)10.1021/acsomega.7b01084 (DOI)000418744100113 ()2-s2.0-85063156989 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180914

Available from: 2018-09-13 Created: 2018-09-13 Last updated: 2020-03-09Bibliographically approved
4. Crystal Structures Combined with Molecular Dynamics Reveal Altered Flow of Water in the Active Site of W60C Chromobacterium violaceum ω-Amino Transaminase
Open this publication in new window or tab >>Crystal Structures Combined with Molecular Dynamics Reveal Altered Flow of Water in the Active Site of W60C Chromobacterium violaceum ω-Amino Transaminase
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-266495 (URN)
Note

QC 20200122

Available from: 2020-01-15 Created: 2020-01-15 Last updated: 2020-01-22Bibliographically approved

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