Protein Complexity via Non-Native States: Binding, Stability, and Structural Studies of Calbindin D9k and HAMLET (Human alpha-lactalbumin Made LEthal to Tumor cells).
(2003)- Abstract
- Using optical spectroscopy, nuclear magnetic resonance (NMR), and differential scanning Calorimetry (DSC), I have studied two different calcium binding proteins that can form kinetically trapped altered states. Calcium is very important in numerous biological processes such as blood coagulation, signal transduction, muscle contraction and bone formation. Calcium binding to proteins regulates these processes. First, the influence of a bound water molecule on the cooperativity of calcium binding in an EF hand protein, calbindin D9k, was studied. Affinity, kinetics, stability and structure properties of two mutant proteins showed the importance of water molecules in the binding process. Surprisingly, one mutant formed a 3D domain swapped... (More)
- Using optical spectroscopy, nuclear magnetic resonance (NMR), and differential scanning Calorimetry (DSC), I have studied two different calcium binding proteins that can form kinetically trapped altered states. Calcium is very important in numerous biological processes such as blood coagulation, signal transduction, muscle contraction and bone formation. Calcium binding to proteins regulates these processes. First, the influence of a bound water molecule on the cooperativity of calcium binding in an EF hand protein, calbindin D9k, was studied. Affinity, kinetics, stability and structure properties of two mutant proteins showed the importance of water molecules in the binding process. Surprisingly, one mutant formed a 3D domain swapped dimer upon crystallization. We showed that packing of a hydrophobic substitution in the linker region is the driving force for formation of the kinetically trapped dimer. The human genome consists of fewer genes than predicted but maintain complexity by mechanisms on different levels from DNA to proteins. In my second project, I studied a system that changes function and structure upon binding a fatty acid ligand. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1, 9 cis) that kills tumor cells by an apoptosis-like mechanism. Previously, it has been shown that only calcium-free, apo, alpha-lactalbumin can be converted to HAMLET. Apo alpha-lactalbumin is partly unfolded. A bovine version, BAMLET and D87A-BAMLET, a converted non-calcium binding, and thus permanently partly unfolded, were both able to kill tumor cells. HAMLET maintained a high affinity for Ca2+ but D87A-BAMLET was active with no Ca2+ bound. The conclusion is that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. The stability towards thermal and urea denaturation was measured for HAMLET, BAMLET and human and bovine alpha-lactalbumin. Three lines of evidence indicate that HAMLET and BAMLET are kinetic traps. I) HAMLET/BAMLET has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid. II) Its denaturation is irreversible and HAMLET/BAMLET is lost after denaturation. III) Formation of HAMLET/BAMLET requires a specific conversion protocol. NMR studies show that oleic acid is bound in a u-shaped fashion in HAMLET, but the spectra of the protein are poorly dispersed further underlining previous observations of a highly dynamic and unstructured state under physiological conditions. (Less)
- Abstract (Swedish)
- Popular Abstract in Swedish
Kalcium är inblandat i många viktiga processer i kroppen. Kalciumbindande proteiner är viktiga i detta sammanhang. Det finns ofta en vattenmolekyl med på bindningsstället i proteinet och vi har studerat hur den påverkar bindningsegenskaperna.
Kalcium är inblandat i många viktiga processer i kroppen. Kalciumbindande proteiner är viktiga i detta sammanhang. Jag har studerat ett protein som agerar som kalciumbuffert i celler, calbindin D9k. Det finns en vattenmolekyl med i kalciumbindningsstället i proteinet och vi har studerat hur den påverkar bindningsegenskaperna.
HAMLET är ett komplex mellan mjölkproteinet alfalaktalbumin och oljesyra. Det dödar cancerceller... (More) - Popular Abstract in Swedish
Kalcium är inblandat i många viktiga processer i kroppen. Kalciumbindande proteiner är viktiga i detta sammanhang. Det finns ofta en vattenmolekyl med på bindningsstället i proteinet och vi har studerat hur den påverkar bindningsegenskaperna.
Kalcium är inblandat i många viktiga processer i kroppen. Kalciumbindande proteiner är viktiga i detta sammanhang. Jag har studerat ett protein som agerar som kalciumbuffert i celler, calbindin D9k. Det finns en vattenmolekyl med i kalciumbindningsstället i proteinet och vi har studerat hur den påverkar bindningsegenskaperna.
HAMLET är ett komplex mellan mjölkproteinet alfalaktalbumin och oljesyra. Det dödar cancerceller med är harmlöst för vanliga celler. Vi har tittat på hur kalciumbindningen ändrats och hur den på verkar bildandet av komplexet och dess anticanceraktivitet. Vidare har vi studerat hur proteinet står emot uppvärmning och en kemikalie som förstör proteiner. Då komplexet är mindre stabilt mot dessa påfrestningar än alfalaktalbumin själv och förlorar sin aktivitet samt blir fri oljesyra och protein så tolkar vi det som att komplexet är en s.k. kinetisk fälla. Ytterligare bevis på detta är att man måste tillverka det enligt en speciell procedur då det inte fungerar att bara blanda oljesyra och protein. Oljesyran har formen av ett U i proteinet och komplexet är mycket rörligare och mer ostrukturerat än det fria proteinet vid normala fysiologiska förhållanden. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/466562
- author
- Fast, Jonas LU
- supervisor
- opponent
-
- Svensson, Birte, Carlsberg research center, Denmark
- organization
- publishing date
- 2003
- type
- Thesis
- publication status
- published
- subject
- keywords
- Biochemistry, apoptosis, oleic acid, HAMLET, kinetic trap alpha-lactalbumin, calbindin, calcium, Metabolism, Biokemi, metabolism
- pages
- 109 pages
- publisher
- Jonas Fast, Biophysical Chemistry Lund University
- defense location
- Room B, Center for Chemistry and Chemical Engineering, Lund Institute of Technology.
- defense date
- 2004-01-30 10:30:00
- ISBN
- 91-628-5934-X
- language
- English
- LU publication?
- yes
- additional info
- Article: An extended hydrophobic core induces EF-hand swapping.Maria Håkansson, Anders Svensson, Jonas Fast and Sara Linse.Protein Science, May 10 (5), 927-933 (2001). Article: Symmetrical Stabilization of Bound Ca2+ Ions in a Cooperative Pair of EF-Hands through Hydrogen Bonding of Coordinating Water Molecules in Calbindin D9k.Jonas Fast, Maria Håkansson, Andreas Muranyi, Garry P. Gippert, Eva Thulin, Johan Evenäs, L. Anders Svensson, and Sara Linse.Biochemistry, 40 (33), 9887-9895 (2001). Article: Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (Human alpha-lactalbumin Made Lethal to tumor cells).Malin Svensson, Jonas Fast, Ann-Kristin Mossberg, Caroline Düringer, Lotta Gustafsson, Oskar Hallgren, Charles L. Brooks, Lawrence Berliner, Sara Linse, and Catharina Svanborg.Protein Science Dec 1(12), 2794-2804 (2003). Article: Stability studies on HAMLET (Human alpha-lactalbumin Made LEthal to Tumor cells): A kinetically trapped alpha-lactalbumin-oleic acid-complex with a novel function.Jonas Fast, Ann-Kristin Mossberg, Catharina Svanborg, and Sara Linse.Submitted to Journal of Molecular Biology. Article: NMR studies of HAMLET (Human alpha-lactalbumin Made LEthal to Tumor cells): A partly folded state kinetically stabilized by oleic acid in a U-shaped conformation.Jonas Fast, Hanna Nilsson, Anna-Christine Mossberg, Catharina Svanborg, Mikael Akke, and Sara Linse.Manuscript
- id
- 423b2409-c92c-4bfb-b316-41c8d42e0b7f (old id 466562)
- date added to LUP
- 2016-04-04 11:51:48
- date last changed
- 2018-11-21 21:07:42
@phdthesis{423b2409-c92c-4bfb-b316-41c8d42e0b7f,
abstract = {{Using optical spectroscopy, nuclear magnetic resonance (NMR), and differential scanning Calorimetry (DSC), I have studied two different calcium binding proteins that can form kinetically trapped altered states. Calcium is very important in numerous biological processes such as blood coagulation, signal transduction, muscle contraction and bone formation. Calcium binding to proteins regulates these processes. First, the influence of a bound water molecule on the cooperativity of calcium binding in an EF hand protein, calbindin D9k, was studied. Affinity, kinetics, stability and structure properties of two mutant proteins showed the importance of water molecules in the binding process. Surprisingly, one mutant formed a 3D domain swapped dimer upon crystallization. We showed that packing of a hydrophobic substitution in the linker region is the driving force for formation of the kinetically trapped dimer. The human genome consists of fewer genes than predicted but maintain complexity by mechanisms on different levels from DNA to proteins. In my second project, I studied a system that changes function and structure upon binding a fatty acid ligand. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1, 9 cis) that kills tumor cells by an apoptosis-like mechanism. Previously, it has been shown that only calcium-free, apo, alpha-lactalbumin can be converted to HAMLET. Apo alpha-lactalbumin is partly unfolded. A bovine version, BAMLET and D87A-BAMLET, a converted non-calcium binding, and thus permanently partly unfolded, were both able to kill tumor cells. HAMLET maintained a high affinity for Ca2+ but D87A-BAMLET was active with no Ca2+ bound. The conclusion is that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. The stability towards thermal and urea denaturation was measured for HAMLET, BAMLET and human and bovine alpha-lactalbumin. Three lines of evidence indicate that HAMLET and BAMLET are kinetic traps. I) HAMLET/BAMLET has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid. II) Its denaturation is irreversible and HAMLET/BAMLET is lost after denaturation. III) Formation of HAMLET/BAMLET requires a specific conversion protocol. NMR studies show that oleic acid is bound in a u-shaped fashion in HAMLET, but the spectra of the protein are poorly dispersed further underlining previous observations of a highly dynamic and unstructured state under physiological conditions.}},
author = {{Fast, Jonas}},
isbn = {{91-628-5934-X}},
keywords = {{Biochemistry; apoptosis; oleic acid; HAMLET; kinetic trap alpha-lactalbumin; calbindin; calcium; Metabolism; Biokemi; metabolism}},
language = {{eng}},
publisher = {{Jonas Fast, Biophysical Chemistry Lund University}},
school = {{Lund University}},
title = {{Protein Complexity via Non-Native States: Binding, Stability, and Structural Studies of Calbindin D9k and HAMLET (Human alpha-lactalbumin Made LEthal to Tumor cells).}},
year = {{2003}},
}