Validation of Protein Dynamics
When a protein binds to another protein, the interface between the two proteins becomes more rigid and stable. This is because the interaction between the two proteins reduces the flexibility of the interface region, making it less susceptible to exchange with deuterium.
By comparing the extent of deuterium exchange in the interface region of a protein bound to its partner protein with that of the unbound protein, we can identify the residues involved in the interaction, as well as regions which have allosteric changes upon binding, and can validate the protein-protein interface.
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Alphafold, a deep learning algorithm developed by researchers at the University of Cambridge, has revolutionized the field of biochemistry by significantly improving our ability to predict the three-dimensional structure of proteins. Before Alphafold, predicting protein structure was a time-consuming and error-prone process, but Alphafold has shown remarkable accuracy and speed in predicting protein structure, making it a valuable tool for researchers in biochemistry and related fields.
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However, the accuracy of Alphafold's predictions is not perfect and it may still produce errors, which could lead to misinterpretations and incorrect conclusions if not carefully validated by experimental methods. We can validate Alphafold structural predictions using HDX. This technique is particularly useful at studying disordered region interfaces.
Select publications involving validation of Protein Dynamics
We have worked with researchers across the globe studying protein dynamics using HDX-MS, including:
Dr. Thomas Leonard from Max Perutz Labs
Reinhardt R, Hirzel K, Link G, Eisler SA, Hägele T, Parson MAH, Burke JE, Hausser A, Leonard TA. (2023). PKD autoinhibition in trans regulates activation loop autophosphorylation in cis. Proc Natl Acad Sci U S A.
Levina A, Fleming KD, Burke JE, Leonard TA. (2022) Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation. Nature Communications.
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Dr. Calvin Yip from University of British Columbia
Udit Dalwadi, Elaina Corrado, Kaelin D Fleming, Brandon E Moeller, Sung-Eun Nam, John E Burke, Calvin K Yip (2022) Biochemical Characterization of the TINTIN Module of the NuA4 Complex Reveals Allosteric Regulation of Nucleosome Interaction. Molecular and Cellular Biology
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Dr. Martha Cyert from Stanford University
.Idil Ulengin-Talkish, Matthew AH Parson, Meredith L Jenkins, Jagoree Roy, Alexis ZL Shih, Nicole St-Denis, Gergo Gulyas, Tamas Balla, Anne-Claude Gingras, Péter Várnai, Elizabeth Conibear, John E Burke, Martha S Cyert. (2021) Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane. Nature communications
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Dr. Michael Airola from Stony Brook Cancer Center
Gu W, Gao S, Wang H, Fleming KD, Hoffmann RM, Yang JW, Patel NM, Choi YM, Burke JE, Reue J, Airola MV (2021) The middle lipin domain adopts a membrane-binding dimeric protein fold. Nature Communications
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Khayyo VI, Hoffmann RM, Wang H, Bell JA, Burke JE, Reue K, Airola MV (2020) Crystal structure of a lipin/Pah phosphatidic acid phosphatase. Nat Commun. (NSERC)
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Dr. Kalle Gehring from McGill
Chen YS, Kozlov G, Armitano J, Moeller BE, Fakih R, Rohaim R, Roux B, Burke JE, Gehring K (2021) Crystal structure of an archaeal CorB magnesium transporter. Nature Communications (NSERC)
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Dr. Raymond Blind from Vanderbilt
Seacrist CD, Kuenze G, Hoffmann RM, Moeller BE, Burke JE, Meiler J, Blind RD (2020) Integrated Structural Modeling of Full-Length LRH-1 Reveals Inter-domain Interactions Contribute to Receptor Structure and Function. Structure.(NSERC)
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Dr. Alan Smrcka from University of Michigan
Fisher IJ, Jenkins ML, Tall GG, Burke JE, Smrcka, AV (2020) ​Activation of Phospholipase C β by Gβγ and Gα q Involves C-Terminal Rearrangement to Release Autoinhibition. Structure.(NSERC)
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Dr. Yusuf Hannun at Stony Brook University
Shanbhogue P, Hoffmann RM, Airola MV, Maini R, Hamelin DJ, Garcia-Diaz M, Burke JE, Hannun YA. (2019) The juxtamembrane linker in neutral sphingomyelinase-2 functions as an intramolecular allosteric switch that activates the enzyme. .J Biol Chem.
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Dr. Jonathan Backer from Albert Einstein College of Medicine
Heitz SD, Hamelin DJ, Hoffmann RM, Greenberg N, Salloum G, Erami Z, Khalil BD, Shymanets A, Steidle EA, Gong GQ, Nürnberg B, Burke JE, Flanagan JU, Bresnick AR, Backer JM. (2019) A Single Discrete Rab5 Binding Site in PI3Kβ is Required for Tumor Cell Invasion. J Biol Chem.
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Dr. Alistair Boraston from University of Victoria
Vickers C, Liu F, Abe K, Salama-Alber O, Jenkins MJ, Springate CMK, Burke JE, Withers SG, Boraston AB (2018) Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-L-fucosidases from GH29. J Biol Chem.
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Dr. Carrie Lucas from Yale University School of Medicine
Takeda AJ, Zhang Y, Dornan GL, Siempelkamp BD, Jenkins ML, Matthews HF, McElwee JJ, Bi W, Seeborg FO, Su HC, Burke JE, Lucas CL. (2017) Novel PIK3CD mutations affecting N-terminal residues of p110δ cause APDS1 in humans. J Allergy Clin Immunol. (NSERC)
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Dr. Martin Boulanger from University of Victoria
Powell CJ, Ramaswamy R, Kelsen A, Hamelin DJ, Warshaw DM, Bosch J, Burke JE, Ward, GE, Boulanger MJ (2018) Structural and mechanistic insights into the function of the unconventional class-XIV myosin, MyoA, from Toxoplasma gondii. Proc Natl Acad Sci U S A.
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Powell CJ, Jenkins ML, Parker ML, Ramaswamy R, Kelsen A, Warshaw DM, Ward GE, Burke JE, Boulanger MJ. Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex. (2017) Journal of Biological Chemistry
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Our own research
Manoj K Rathinaswamy, Udit Dalwadi, Kaelin D Fleming, Carson Adams, Jordan TB Stariha, Els Pardon, Minkyung Baek, Oscar Vadas, Frank DiMaio, Jan Steyaert, Scott D Hansen, Calvin K Yip, John E Burke (2021) Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation. Science Advances
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Rathinaswamy MK, Jenkins ML, Duewell BR, Zhang X, Harris NJ, Evans JT, Stariha JTB, Dalwadi U, Fleming KD, Ranga-Prasad H, Yip CK, Williams RL, Hansen SD, Burke JE. (2023). Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs. Cell Reports.
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Harris NJ, Jenkins ML, Dalwadi U, Fleming KD, Nam SE, Parson MAH, Yip CK, Burke JE (2021) Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex. Journal of Molecular Biology (NSERC)
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Burke J.E, Inglis, A.J., Perisic, O.,Masson, G.R., Mclaughlin, S., Rutaganira, F., Shokat, K.M., Williams, R.L., 2014. Structures of PI4KIIIß complexes reveal a mechanism for simultaneous recruitment of Rab11 and its effectors. Science. 344(6187), 1035-8.
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Fowler ML, McPhail JA, Jenkins ML, Masson GR, Rutaganira FU, Shokat KM, Williams RL, Burke JE. (2016). Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11. Protein Sci.
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Rathinaswamy MK, Gaieb Z, Fleming KD, Borsari C, Harris NJ, Moeller BJ, Wymann MP, Amaro RE, Burke JE* (2021) Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveals a path to design selective inhibitors. Elife. 10:e64691
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Jenkins ML, Harris NJ, Dalwadi U, Fleming KD, Ziemianowicz​ DS, Rafiei A, Martin EM, Schriemer DC, Yip CK, Burke JE (2020) The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity. Communication Biology.
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Jenkins ML , Ranga-Prasad H, Parson MAH, Harris NJ , Rathinaswamy MK, Burke JE. (2023). Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus. Nature Communications
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Dornan GD, Stariha JTD, Rathinaswamy MK, Powell CJ, Boulanger MJ, Burke JE (2019) Defining how oncogenic and developmental mutations of PIK3R1 alter the regulation of class IA phosphoinositide 3-kinases. Structure.
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McPhail JA, Lyoo HR, Pemberton JG, Hoffmann RH, van Elst W, Strating JRPM, Jenkins ML, Stariha JTB, Balla T*, van Kuppeveld FJM*, Burke JE* (2019) Characterization of the Golgi c10orf76-PI4KB complex, and its necessity for Golgi PI4P levels and enterovirus replication. EMBO reports.
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Thibodeau MC, Harris NJ, Jenkins ML, Parson MAH, Evans JT, Scott MK, Shaw AL, Pokorný D, Leonard TA, Burke JE (2023) Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11. Journal of Biological Chemistry
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Jenkins ML, Margaria JP, Stariha JTB, Hoffmann RM, McPhail JA, Hamelin DJ, Boulanger MJ, Hirsch E, Burke JE. Structural determinants of Rab11 activation by the guanine nucleotide exchange factor SH3BP5. (2018) Nature Communications.
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Dornan GL, Dalwadi U, Hamelin DJ, Hoffmann RM, Yip CK, Burke JE. (2018). Probing the Architecture, Dynamics, and Inhibition of the PI4KIIIα/TTC7/FAM126 Complex. J Mol Biol.
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Siempelkamp BD, Rathinaswamy MK, Jenkins ML, Burke JE. (2017). Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas. J Biol Chem.
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McPhail JA, Ottosen EH, Jenkins ML, Burke JE. (2017). The Molecular Basis of Aichi Virus 3A Protein Activation of Phosphatidylinositol 4 Kinase IIIβ, PI4KB, through ACBD3. Structure. (CIHR)
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Dornan, GD, Siempelkamp, BD, Jenkins, ML, Vadas, O, Lucas, CL, Burke, JE. (2017). Conformational disruption of PI3Kδ regulation by primary immunodeficiency mutations in PIK3CD and PIK3R1. Proc Natl Acad Sci U S A.