Allosteric Regulation CURES
Non Covalent Interactions, Subunit Interactions and Regulation of MDH
Research project this CURE supports: Allosteric Regulation
Description of the Science/background for this CURE:
Many oligomeric enzymes show non canonical homotropic enzyme kinetic or ligand binding behaviour that is important in regulation of their activity, however the simple existence of such non canonical behaviour is not sufficient to involve allosteric regulation of subunit cooperativity as there are many other explanations possible. Likewise, heterotropic non covalent regulation may be allosteric but not involve subunit cooperativity or interactions which involves the transmission of information from one subunit to another in an oligomeric structure via conformational changes. Pioneering work by Bell and Dalziel established a variety of ways to detect such changes and combined with modern computational approaches paved the way to explore the mechanism of such subunit interactions. Various Malate Dehydrogenases show a variety of non-canonical kinetic or ligand binding data and a reciprocating subunit model for both catalytic steps and regulation has been proposed but not substantiated.
Relevant Literature that support this science:
General Background to Malate Dehydrogenase
Literature sources that support this science:
Bell, E & Bell J , “Allosterism and Drug Discovery”., Burger’s Medicinal Chemistry, Drug Discovery and Development, Eighth Edition. Volume 2, pages 163-240, 2021. Publisher- Wiley
Ellis Bell, Proteins & Enzymes, Chapters 12 (Conformational Changes), 16 (Deviations from Linear Kinetics) & 21(Allosteric Models of Enzyme Regulation) , Ellis Bell.
Bell JE, Dalziel K. A conformational transition of the oligomer of glutamate dehydrogenase induced by half-saturation with NAD + or NADP + . Biochim Biophys Acta. 1973 May 5;309(1):237-42. doi: 10.1016/0005-2744(73)90336-7. PMID: 4145351.
Sinha S, Tam B, Wang SM. Applications of Molecular Dynamics Simulation in Protein Study. Membranes (Basel). 2022 Aug 29;12(9):844. doi: 10.3390/membranes12090844. PMID: 36135863; PMCID: PMC9505860.
M. Karplus and J. Kuriyan., Molecular dynamics and protein function., May 3, 2005, 102 (19) 6679-6685
https://doi.org/10.1073/pnas.040893010
Harada K, Wolfe RG. Malic dehydrogenase. VII. The catalytic mechanism and possible role of identical protein subunits. J Biol Chem. 1968 Aug 10;243(15):4131-7. PMID: 4299102.
Harada K, Wolfe RG. Malic dehydrogenase. VI. A kinetic study of hydroxymalonate inhibition. J Biol Chem. 1968 Aug 10;243(15):4123-30. PMID: 5666952.
Michael Schwabe, Ellis Bell, Jessica Bell., Using ANS to Probe Ligand Induced Conformational States of MalateDehydrogenase
First published: 01 April 2016
https://doi.org/10.1096/fasebj.30.1_supplement.600.19
Jacqunae Mays, James Marion, Ellis Bell., The Effects of Citrate on Glyoxasomal Malate Dehydrogenase
First published: 01 April 2012
https://doi.org/10.1096/fasebj.26.1_supplement.558.3
Jimmy Marion, Ellis Bell., The Regulation of Malate Dehydrogenase by Citrate
First published: 01 April 2009
https://doi.org/10.1096/fasebj.23.1_supplement.676.10
John Abano, Ellis Bell, Jessica Bell., The Role of Subunit Interfaces of Malate Dehydrogenase in Protein Folding &Unfolding
First published: 01 April 2016
https://doi.org/10.1096/fasebj.30.1_supplement.600.18
Nikia James, Jayme Verdi, Ellis Bell, Jessica Bell., Probing the Subunit Interface of Malate Dehydrogenase: Effects of S266A andL269A Mutations
First published: 01 April 2016
https://doi.org/10.1096/fasebj.30.1_supplement.600.21
Jacqunae Mays, Ellis Bell., Allosteric Regulation of Glyoxasomal Malate Dehydrogenase by Citrate InvolvesReciprocating Active Site Communication.
First published: 01 April 2013
https://doi.org/10.1096/fasebj.27.1_supplement.789.10
Adam Johnson, Ellis Bell., Engineering Tryptophan Residues into Glyoxysomal Malate Dehydrogenase asProbes of Structure and Function
First published: 06 March 2006
https://doi.org/10.1096/fasebj.20.4.A53-b
Zachary Stewart, Nina Marie Garcia, Ellis Bell, Jessica Bell., The Effects of the H90 and E256 Mutations in the gMDH Interface on the Stability,Function, and Interface Interactions of gMDH
First published: 01 April 2016
https://doi.org/10.1096/fasebj.30.1_supplement.600.2
Nina Marie Garcia, Michael Schwabe, Sasha Graham, Ellis Bell., Probing the role of the interface on activity and regulation of gMDH
First published: 01 April 2018
https://doi.org/10.1096/fasebj.2018.32.1_supplement.528.8
3-5 Learning goals for this CURE:
1. Students will appreciate that a good research project entails nine essential elements of research and will develop a novel hypothesis that makes predictions that can be tested experimentally, and present a proposal for their project. Rubric 1
2. Students will learn how to design and execute experiments to test their hypothesis, will learn appropriate data analysis approaches and will appreciate the importance of accurate documentation of their work and reproducibility of their experiments. Rubric 2
3. Students will learn to develop a description of their research project in written, poster or a slide presentation suitable for verbal presentation. Rubric 3
Research question for this CURE:
1. Can you develop a way to demonstrate that non canonical behaviour in malate dehydrogenase is due to subunit interactions
2. Can you understand the target structure-function relationships that underpin potential allosteric interactions in malate dehydrogenase?
3. Can you propose and initiate potential strategies to identify the triggers, information relay path and elicitors of intersubunit communication in malate dehydrogenase?
2-3 Sample hypotheses students could come up with for this CURE:
Introductory lectures have defined types of approaches to drug development and can be broken down to two basic approaches: structure based drug design or a screening potential candidates approach. Students can select one of these approaches or the approach can be set by the instructor.
STUDENTS ARE LED THROUGH HYPOTHESIS AND PROPOSAL PREPARATION USING THE RULE OF THREES APPROACH:
Typically student hypotheses hone in on some unique aspect of the subunit interface structure (established by comparative Hawkdock Analysis), or some key aspects of the ligand binding site depending on the overall question they choose to study.
What Experimental Approaches can be Incorporated?
WET LAB TECHNIQUES: LINK TO THEORY AND PRACTICAL DESCRIPTIONS OF WET LAB TECHNIQUES
Computational Techniques
CURE FORMAT: MODULAR, SEMESTER, EITHER
Week by week lab activities for a modular version (6 weeks) and/or semester long version of this CURE :
The CURE starts with discussions of the target enzyme, Malate Dehydrogenase and its roles in metabolism. This is followed by details of the reaction catalyzed and the role that identical subunits in an oligomer might play- ie subunit interactions could be involved in catalysis or in regulation. After appropriate literature review and bioinformatics student groups decide on a relevance angle and what basic science issue they want to explore, focusing on either the subunit interface and transimision of information across the interface of on the active site region and what interactions occuring in the active site trigger the relay of information to the interface or from the interface to an adjacent active site. Students develop a hypothesis, and as appropriate make the appropriate mutants (or select from existing mutants), express and purify proteins (mutant and wild type) and conduct the various experiments they propose to explore their hypothesis.
Ideal group size for this CURE: Groups of 2-3 students
Ideal course/level for this CURE (chem, bio, biochem, interdisciplinary; first year, middle years, capstone): (list as many as are possibilities)
middle years, upper level
Teaching Resources Available:
Powerpoints to Introduce Each Weeks Lab Session
Generic Customizable Templates for student activities for each of the 9 essential elements of research incorporated into the CURE
T4: Proposal
T5: Experimental Design & Execution
T6: Reproducibility
T7: Data Analysis & Conclusions
Rubrics to Guide & Assess Student Performance
Instrumentation/equipment/key reagents needed for this CURE
Uv-vis Spectrophotometer, Equipment for acid-base titrations, pH Meter, Balance, Water Bath, Stir Plate
Protein (WT and/or specific mutant), organism:
Plasmids needed can be obtained from Addgene:
Plasmodium falciparum, Human Mitochondrial, Human Cytosolic
Clone Data Sheets for this Project Area: While many students choose to work with watermelon glyoxysomal MDH, in reality any of the wild type isoforms available could be used.
Watermelon Glyoxysomal Malate Dehydrogenase
Ignicoccus Islandicus Malate Dehydrognease
MW(subunit/biological)/pI/ e280 , extinction coefficient (280 nm: calculated using ProtParam.) of protein (WT and/or specific mutant):
Plasmodium falciparum: MWt: 35,715/142,860, pI(theoretical): 6.89 e280 0.375 mL.mg-1.cm-1
Human Mitochondrial: MWt: 34,806/69612, pI(theoretical): 8.33 e280 0.257 mL.mg-1.cm-1
Human Cytosolic:MWt: 39,749/79,498, pI(theoretical): 7.14 e280 0.853 mL.mg-1.cm-1
PDB ID for the WT version of these protein :
Plasmodium falciparum: 5.nfr.pdb
Human Cytosolic: 7rm9.pdb
Human Mitochondrial: 2dfd.pdb
Available Resources for structural analysis and computational approaches: Biologically relevant pdb files
Plasmodium falciparum Tetramer
Human Cytosolic Dimer
Human Mitochondrial Dimer
Landmarked .pse files for use with the project - see clone datasheets for descriptions