Skip to main content

Departments

J1-4400 Evaluation of transient protein states

Project summary:

Despite recent advances in biophysical techniques and computational approaches for the investigation of biomolecular systems, the characterization of the dynamic equilibria of protein states remains a demanding challenge due to the elusive nature of the dynamic processes that regulate these equilibria. In particular, the sparsely populated, transient states are difficult to characterize at the atomic level because they are "invisible" to the biophysical methods that detect protein states with the lowest energy or the highest abundance. There is growing evidence for the importance of transient, thermally accessible protein states in the biological function of proteins.

The main goal of the proposed project is to develop a combined experimental and theoretical approach for the physicochemical characterization at the atomic level of transient protein states close to physiological conditions in aqueous environments. The characterization will allow to relate them to the specific dynamic event that is important for their function. To overcome the problems of averaging and signal assignment, a combination of solution NMR and vibrational spectroscopy operating on different time scales will be used. Together, these spectroscopies can provide access to transient molecular states in dynamic exchange on time scales from picoseconds to milliseconds. To obtain a model-based understanding of the correlation between each type of motion and its effect on dynamic equilibria of states, we will use atomistic molecular dynamic simulations. Algorithms for numerical analysis of the correlation between experimental and computational results will be developed to understand the influence of the individual contributions of the coupled motions on the distribution of protein states. Proteins of different families will be studied, covering a wide range of dynamic events that can cause transitions between thermally accessible protein states. The functional role of the selected proteins in biological processes cannot be comprehensively understood without examining the dynamic equilibria between their possible states without and/or with their binding partners. The expected results may provide presently unforeseen insights into the function of these proteins in health and disease. 

The developed approach will be widely applicable to the characterization of transient protein states at the atomic level in aqueous environments. The results of the proposed studies can contribute significantly to the understanding of protein function in health and disease and will be of great interest to many specific research areas, such as molecular recognition, molecular signal transduction, enzyme catalysis, protein folding and binding, and target-based design and drug discovery.

 

Participating organizations: 

  • University of Ljubljana, Faculty of Pharmacy

            a. basic information regarding financing

The project is co-financed by ARRS with 2481 annual hours of price class B for a period of 3 years. Funding starts on October 1, 2022.

            b. composition of the project group with links to SICRIS

At the National Institute of Chemistry, the project group includes:

Simona Golič Grdadolnik; SICRIS št. 08329;

Franci Merzel; SICRIS št. 13627

Jože Grdadolnik; SICRIS št. 08523

Tjaša Goričan; SICRIS št. 39078

Barbara Zupančič; SICRIS št. 28608

Gregor Anderluh; SICRIS št. 15686

Iza Ogris; SICRIS št. 50459

Neli Sedej; SICRIS št. 54913

Aljoša Marinko; SICRIS št. 57301

At the Faculty of Pharmacy (University of Ljubljana), the project group includes:

Stanislav Gobec; SICRIS št. 15284

Tihomir Tomašić; SICRIS št. 28334

Izidor Sosič; SICRIS št. 30816

Martina Hrast Rambaher; SICRIS št. 32036

Živa Zajec; SICRIS št. 53670

 

Phases of the project and their realization

The work is organized into three work packages (WP). 

WP1: Identification and evaluation of transient conformational states of aegerolysins with intrinsically disordered regions.

Due to the lower stability of the phiatt and fusver proteins, the outlined studies were first performed on the more stable protein PpNPP1, which allows a broader range of NMR experiments on the isotopically labelled protein and also has ordered and disordered regions that may be important for interaction with lipids. The knowledge gained allowed us to determine a set of parameters suitable for performing NMR experiments on less stable proteins. We assigned the chemical shifts of the main chain and side chains of PpNPP1 and determined the structure of the ordered part of the protein in solution. We also determined the interaction of different lipids with PpNPP1 using NMR methods. We identified two classes of exchange between the conformational states of the disordered part of PpNPP1 from a few hundred milliseconds to one second and from micro- to milliseconds, which could influence lipid binding. 15N NMR relaxation experiments on Fusver, Phiatt and PpNPP1 proteins have been partially performed. We have performed a biophysical characterization of the activity and lipid interactions of Fusver, Phiatt and PpNPP1 proteins.

WP2: Identification and evaluation of transient conformational states of MurD related to substrate and inhibitor binding.

We have successfully prepared complex isotopically labelled MurD in two different isotopic labelling schemes and performed NMR measurements to assign the AILVproR methyl groups. These are 3D-methyl-methyl-NOESY for the assignment of methyl groups via spatial proximity on (U)-2H15N-selectively-13CH3-isotopically labelled MurD and 3D-out-and-back-13C-TOSCY and 3D-BEST-HNCA for the complementary assignment of methyl groups via the main chain of the protein on (U)-2H-13C-15N- selectively-13CH3 isotopically labelled MurD. We performed extensive molecular dynamics simulations of MurD alone and eleven complexes of MurD with substrates and inhibitors. Sparsely populated states were present in all simulations, and among the ground states we also identified a very closed conformation of MurD that has not been observed experimentally so far. In the further course of the project, we will use NMR methods to experimentally describe different conformational state of MurD via NMR parameters of methyl groups. By measuring the NMR relaxation parameters of the MurD backbone and determining the spectral density to characterize the frequency spectrum of the moitions of the N-H vectors, we have already established that at certain regions of the backbone an exchange between conformational states takes place on a time scale of micro- to milliseconds, which is typical for the exchange between basic and higher-energy transition states. An exchange on this time scale was observed both in free MurD and in complexes with substrate and inhibitor.

WP3: Identification and evaluation of transient conformational states of NGF related to the binding of small endogenous ligands.

Using an integrative structural biophysics analysis, we have identified and defined the binding of ATP and divalent ions to rhNGF and its precursor rh-proNGF at the atomic level and determined their influence on the interaction with receptors. We show that ATP binds to the intrinsically disordered propeptide rh-proNGF with a micromolar dissociation constant, in contrast to the weak millimolar binding of ATP to rhNGF. We found that ATP binding triggers a conformational rearrangement of the flexible propeptide domain of rh-proNGF, suggesting that ATP acts as an allosteric modulator of the overall conformation of rh-proNGF. We demonstrated that Mg2+, which plays a known physiological role in neurons, modulates the ATP/proNGF interaction. We continue to define the interaction of endogenous lipids with rhNGF.

Bibliographic references that derive directly from the implementation of the project

Original scientific articles:

1. PAOLETTI, Francesca, COVACEUSZACH, Sonia, CASSETTA, Alberto, CALABRESE, Antonio N., NOVAK, Urban, KONAREV, Peter, GRDADOLNIK, Jože, LAMBA, Doriano, GOLIČ GRDADOLNIK, Simona. Distinct conformational changes occur within the intrinsically unstructured pro-domain of pro-Nerve Growth Factor in the presence of ATP and Mg2+. Protein science. vol. 32, iss. 2, e4563, str. 1-14, ilustr. ISSN 1469-896X. https://onlinelibrary.wiley.com/doi/epdf/10.1002/pro.4563DiRROS - Digitalni repozitorij raziskovalnih organizacij Slovenije, DOI: 10.1002/pro.4563. [COBISS.SI-ID 139671043]

2. DIAMANTIS, Dimitrios A., TSIAILANIS, Antonios D., PAPAEMMANOUIL, Christina, NIKA, Maria-Christina, KANAKI, Zoi, GOLIČ GRDADOLNIK, Simona, BABIČ, Andrej, TZAKOS, Eleftherios Paraskevas, FOURNIER, Isabelle, SALZET, Michel, KUSHWAHA, Prem Prakash, THOMAIDIS, Nikolaos S., RAMPIAS, Theodoros, SHANKAR, Eswar, KARAKURT, Serdar, GUPTA, Sanjay Kumar, TZAKOS, Andreas G. Development of a novel apigenin prodrug programmed for alkaline-phosphatase instructed self-inhibition to combat cancer. Journal of biomolecular structure & dynamics. 28. Aug. 2023, str. 1-22, https://www.tandfonline.com/doi/epdf/10.1080/07391102.2023.2247083?needAccess=true&role=buttonDOI: 10.1080/07391102.2023.2247083. [COBISS.SI-ID 163337219]

Presentations at conferences:

1. PAOLETTI, Francesca, MERZEL, Franci, COVACEUSZACH, Sonia, CASSETTA, Alberto, CALABRESE, Antonio N., OGRIS, Iza, NOVAK, Urban, KONAREV, Peter, GRDADOLNIK, Jože, LAMBA, Doriano, GOLIČ GRDADOLNIK, Simona. Differential interactions between ATP and NGF / proNGF: chance or necessity? V: 62° congress SIB : 7-9 September [2023], Firenze : abstract book. [S. l.: s. n.], 2023. Str. 42. https://www.congressosib2023.it/wp-content/uploads/2023/09/Abstract-Book_CongressoSIB-2023.pdfhttps://www.congressosib2023.it/abstract-book/. [COBISS.SI-ID 176813315]

2. ŽIBRAT, Nika, PODOBNIK, Marjetka, GOLIČ GRDADOLNIK, Simona, ANDERLUH, Gregor. Phytophthora parasitica NLP shares three-dimensional fold and pore-forming activity of model Nep1-like protein, NLPPya. V: SEPČIĆ, Kristina (ur.), PAVŠIČ, Miha (ur.). 15th Meeting of the Slovenian Biochemical Society with International Participation = 15. srečanje Slovenskega biokemijskega društva z mednarodno udeležbo : book of abstracts = zbornik povzetkov : Portorož, 20-23 September 2023 = [20.–23. september 2023, Portorož, Slovenija]. Ljubljana: Slovenian Biochemical Society: = Slovensko biokemijsko društvo, 2023. Str. 171. ISBN 978-961-95941-1-7. https://portoroz2023.sbd.si/upload/docs/SBD2023_Portoroz-Book_of_Abstracts.pdf. [COBISS.SI-ID 170124035]
3. HERLAH, Barbara, OGRIS, Iza, GOLIČ GRDADOLNIK, Simona, SOSIČ, Izidor, GOBEC, Stanislav, PERDIH, Andrej. Simulation-based optimization of 4,6-substituted-1,3,5-triazin-2(1H)-ones as catalytic inhibitors of the human DNA Topoisomerase IIΑ. V: Abstracts book : Lisbon 2023, at the edge of Europe and Science. [S. l.]: [s. n.], 2023. Str. 88. https://annualmeeting.eufeps.org/programme.html. [COBISS.SI-ID 156434691].

4. DERNOVŠEK, Jaka, GORIČAN, Tjaša, ZAJEC, Živa, ZIDAR, Nace, ILAŠ, Janez, URBANČIČ, Dunja, ZUBRIENÉ, Asta, MATULIS, Daumantas, GOLIČ GRDADOLNIK, Simona, TOMAŠIČ, Tihomir. Development of new HSP90β-selective inhibitors based on topoisomerase II ligands. V: JAKOPIN, Žiga (ur.), TOMAŠIČ, Tihomir (ur.). Crossing the Boundaries of Medicinal Chemistry: book of abstracts : University of Ljubljana, Faculty of Pharmacy, September 28, 2023. Ljubljana: Fakulteta za farmacijo, 2023. Str. 21, ilustr. ISBN 978-961-6378-95-6. https://www.ffa.uni-lj.si/docs/default-source/e-knjige/book-of-abstracts---crossing-the-boundaries-of-medicinal-chemistry.pdf?sfvrsn=2. [COBISS.SI-ID 173421059]
5. HERLAH, Barbara, OGRIS, Iza, GOLIČ GRDADOLNIK, Simona, GOBEC, Stanislav, SOSIČ, Izidor, PERDIH, Andrej. Structure-based optimization of 4,6-substituted-1,3,5-triazin2(1H)-ones as catalytic inhibitors of human DNA topoisomerase IIα. V: PINTAR, Albin (ur.). Slovenski kemijski dnevi 2023 = 29th Annual Meeting of the Slovenian Chemical Society : zbornik povzetkov = book of abstracts : 13.-15. september 2023, Portorož, Portorose, Slovenija. Ljubljana: Slovensko kemijsko društvo, 2023. Str. 118. ISBN 978-961-95922-2-9. https://www.conftool.org/scs-annual-meeting-2023/index.php?page=browseSessions&form_session=47#paperID119https://www.conftool.org/scs-annual-meeting-2023/index.php/Herlah-Structure-based_optimization_of_4%2C6-substituted-1%2C3%2C5-triazin-2-119.pdf?page=downloadPaper&filename=Herlah-Structure-based_optimization_of_4%2C6-substituted-1%2C3%2C5-triazin-2-119.pdf&form_id=119. [COBISS.SI-ID 167932931]

Accessibility(CTRL+F2)
color contrast
text size
highlighting content
zoom in