Crystallization, optimization, and crystal structure determination of ligands in complex with galectin-8N
(2022) KEMK10 20221Department of Chemistry
- Abstract
- This thesis focuses on the protein “galectin-8N” which is a lectin protein that takes part in several physiological functions such as inflammation, autophagy, cell migration, and more. The project has three objectives: to observe and increase the reproducibility of crystallization of two variants (wildtype and mutant) of the human protein galectin-8N and to analyze the binding site when three different novel ligands are each inserted. Lastly, attempts of crystallizing mouse galectin-8N was also investigated which there is no prior crystallization data of.
To observe required conditions for crystallizing and to increase the reproducibility of the two variants of human galectin-8N, two commercial plates of “MorpheusTM” were used and four... (More) - This thesis focuses on the protein “galectin-8N” which is a lectin protein that takes part in several physiological functions such as inflammation, autophagy, cell migration, and more. The project has three objectives: to observe and increase the reproducibility of crystallization of two variants (wildtype and mutant) of the human protein galectin-8N and to analyze the binding site when three different novel ligands are each inserted. Lastly, attempts of crystallizing mouse galectin-8N was also investigated which there is no prior crystallization data of.
To observe required conditions for crystallizing and to increase the reproducibility of the two variants of human galectin-8N, two commercial plates of “MorpheusTM” were used and four manual plates imitating the conditions of Morpheus. After crystals were observed in the droplets of the manual plates, one of the plates was used to soak the ligand MH-3-80 and the other two to cocrystallize with ligands MH-3-80, MH-1FH and NV9.1. All the produced crystals were brought to the MAX IV where data was collected from and through iterative refinement processes with phenix, five models of human galectin-8N were created. From the models, all interactions between inserted ligands and the binding site were observed via PyMOL. Lastly, crystallization attempts of mouse galectin-8N were experimented on in total of eight commercial screens, two buffer screens, and one Morpheus screen combined with “Seed Beads”. The results were that both variants of human galectin-8N crystallizes in the Morpheus commercial plates and well in manual screens imitating Morpheus’ conditions. Crystals were found to prefer conditions of: buffer 1, in precipitant mixture 4, and with additives such as ethylene glycols, monosaccharides and alcohols. Ligands MH-3-80 and MH-1FH were inserted successfully into both variants of the human proteins, while NV9.1 did not. The interactions that occurred between inserted ligands and the residues at the binding site were: Arg52, His72, Arg76, Asn86, and Trp93. Other interactions were also observed however those existed exclusively depending on the inserted ligands’ structure and their moieties. Lastly, the crystallization of mouse galectin-8N was unfortunately difficult and rather unsuccessful, however the commercial screen: JCSG+TM gave the most promising results and is highly recommended for future observations. (Less) - Popular Abstract (Swedish)
- Studien fokuserade på proteinet “galectin-8N”, vilket är ett lectinprotein av betydelse för vissa kroppsliga funktioner som till exempel inflammation, autofagi och cellmigration. Arbetet omfattade tre delmål: 1. att kristallisera två varianter av mänskligt galectin-8N och öka dess reproducerbarhet; 2. att studera bindningar och interaktioner mellan bindningsställen för humana proteinerna och tre stycken ligander (en molekyl som binder), samt 3. att försöka kristallisera mus galectin-8N, vilket ingen har lyckats med tidigare. Studien började med att observera kristalliseringstillstånden för galectin-8N genom att använda en “MorpheusTM” platta, vilket är en metod som används för att undersöka protein där kristalliseringstillstånden är... (More)
- Studien fokuserade på proteinet “galectin-8N”, vilket är ett lectinprotein av betydelse för vissa kroppsliga funktioner som till exempel inflammation, autofagi och cellmigration. Arbetet omfattade tre delmål: 1. att kristallisera två varianter av mänskligt galectin-8N och öka dess reproducerbarhet; 2. att studera bindningar och interaktioner mellan bindningsställen för humana proteinerna och tre stycken ligander (en molekyl som binder), samt 3. att försöka kristallisera mus galectin-8N, vilket ingen har lyckats med tidigare. Studien började med att observera kristalliseringstillstånden för galectin-8N genom att använda en “MorpheusTM” platta, vilket är en metod som används för att undersöka protein där kristalliseringstillstånden är okända. Utifrån den data gjordes nya manuella plattor för att simulera Morpheus-tillstånden och öka deras reproducerbarhet. Tre ligander: MH-3-80, MH-1FH och NV9.1 fördes in till bindningsstället genom att använda teknikerna “ligandblötläggning” eller “cokristallisation“ manuellt i nya plattor. Kristallerna som formades samlades upp och analyserades i MAX IV. Därefter gjordes en förfining av modellerna som möjliggjorde observationer av interaktionen mellan liganderna och bindningsstället. Försökte gjordes att kristallisera musproteinet genom att använda flera kommersiella plattor likt “MorpheusTM” och dessutom observerades dess termiska stabilitet i olika buffrar. “Morpheus” plattorna resulterade i garanterade kristaller och optimering (simulering) producerade kristaller av mänskligt galectin-8N. Reproducerbarheten var bra och fungerade med hög säkerhet. Liganderna MH-3-80 och MH-1FH kunde föras in i båda varianterna av mänskligt galectin-8N, medan NV9.1 misslyckades. Efter observation i datorprogrammet PyMOL var det möjligt att se att aminosyrorna Arg52, His72, Arg76, Asn86 och Trp93 alltid hade interaktion mellan ligand och bindningsställe. Det var dessutom möjligt att observera vissa exklusiva interaktioner som berodde på ligand-molekylens struktur. För musproteinet var det svårt att bedöma om kristaller hade bildats eller om det var proteinklumpar; majoriteten av experimenten misslyckades. Bästa resultaten observerades för plattan kallad “JCSG+”. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9088868
- author
- Larsson, George LU
- supervisor
-
- Derek Logan LU
- organization
- course
- KEMK10 20221
- year
- 2022
- type
- M2 - Bachelor Degree
- subject
- keywords
- Biochemistry, Structural Biochemistry, Galectin-8, Protein Crystallization
- language
- English
- id
- 9088868
- date added to LUP
- 2022-06-30 09:26:45
- date last changed
- 2022-06-30 09:26:45
@misc{9088868,
abstract = {{This thesis focuses on the protein “galectin-8N” which is a lectin protein that takes part in several physiological functions such as inflammation, autophagy, cell migration, and more. The project has three objectives: to observe and increase the reproducibility of crystallization of two variants (wildtype and mutant) of the human protein galectin-8N and to analyze the binding site when three different novel ligands are each inserted. Lastly, attempts of crystallizing mouse galectin-8N was also investigated which there is no prior crystallization data of.
To observe required conditions for crystallizing and to increase the reproducibility of the two variants of human galectin-8N, two commercial plates of “MorpheusTM” were used and four manual plates imitating the conditions of Morpheus. After crystals were observed in the droplets of the manual plates, one of the plates was used to soak the ligand MH-3-80 and the other two to cocrystallize with ligands MH-3-80, MH-1FH and NV9.1. All the produced crystals were brought to the MAX IV where data was collected from and through iterative refinement processes with phenix, five models of human galectin-8N were created. From the models, all interactions between inserted ligands and the binding site were observed via PyMOL. Lastly, crystallization attempts of mouse galectin-8N were experimented on in total of eight commercial screens, two buffer screens, and one Morpheus screen combined with “Seed Beads”. The results were that both variants of human galectin-8N crystallizes in the Morpheus commercial plates and well in manual screens imitating Morpheus’ conditions. Crystals were found to prefer conditions of: buffer 1, in precipitant mixture 4, and with additives such as ethylene glycols, monosaccharides and alcohols. Ligands MH-3-80 and MH-1FH were inserted successfully into both variants of the human proteins, while NV9.1 did not. The interactions that occurred between inserted ligands and the residues at the binding site were: Arg52, His72, Arg76, Asn86, and Trp93. Other interactions were also observed however those existed exclusively depending on the inserted ligands’ structure and their moieties. Lastly, the crystallization of mouse galectin-8N was unfortunately difficult and rather unsuccessful, however the commercial screen: JCSG+TM gave the most promising results and is highly recommended for future observations.}},
author = {{Larsson, George}},
language = {{eng}},
note = {{Student Paper}},
title = {{Crystallization, optimization, and crystal structure determination of ligands in complex with galectin-8N}},
year = {{2022}},
}