Structural biology of Functional and Pathological Amyloids
We use modern NMR spectroscopy to study functional and pathological amyloid fibrils. The focus of research for the functional amyloid topic is biofilm forming functional bacterial amyloid fibrils (FuBA). Our functional amyloid systems are a unique class of amyloid fibrils with specific biological functions in living organisms, i.e., bacteria, in contrast to the pathological amyloids causing disease as neurodegenerative ones (αS or Aβ). A subgroup of these systems is bacterial biofilm forming FuBAs (such as CsgA, FapC, TasA or PSMs), a major cause of persistent infections and an antimicrobial resistance (AMR) target. We focus on ssNMR-based structural biology of these biofilm forming functional amyloids. We aim to determine atomic resolution structure and molecular dynamics information, for better understanding of amyloid formation, biofilms, and their interactions. This will pave the way towards future treatments against bacterial infections and their antimicrobial resistance. In my group we also utilize solution-state NMR spectroscopy to study the prefibrillar soluble monomeric FapC and also on pilin ComGC, that is an intrinsically disordered protein (IPD) and its interaction with other proteins within the same biofilm forming protein operon. Particularly, the IDP chaperone-like FapA interaction with FapC is focused, and we recently showed that FapA remarkably slows down the FapC fibril formation without modifying the final fibril morphology. Our studies will establish a better understanding of the functional amyloid formation in bacterial biofilms and its molecular level tuning or prevention by chaperones. We foresee that future therapeutic treatments of biofilms and relevant infections could emerged from these structural and mechanistic insights.
This research topic is in close collaboration with Prof. James Conway from University of Pittsburgh, USA, Prof. Maria Andreasen and Poul Henning Jensen from Aarhus University, Denmark, and Prof. Guido Pintacuda from ENS Lyon, France.
Chang-Hyeock Byeon, Kasper Holst Hansen, Jasper Jeffrey, Hakan Saricayir, Maria Andreasen, Ümit Akbey. Intrinsically disordered Pseudomonas chaperone FapA slows down the fibrillation of major biofilm-forming functional amyloid FapC. The FEBS Journal, 2024. DOI: doi.org/10.1111/febs.17084
Chang-Hyeock Byeon, Pang C. Wang, In-Ja L. Byeon, Ümit Akbey. Solution-state NMR assignment and secondary structure propensity of the full length and minimalistic-truncated prefibrillar monomeric form of biofilm forming functional amyloid FapC from Pseudomonas aeruginosa. BiomolNMRAssign, 2023. https://doi.org/10.1007/s12104-023-10135-5
Ümit Akbey, Maria Andreasen. Functional amyloids from bacterial biofilms – structural properties and interaction partners. Invited Review. RSC – Chemical Science, 13(22), 6457-6477, 2022. DOI: 10.1039/d2sc00645f
Nelson Ferreira, Emil Gregersen, Zachary A. Sorrentino, Hjalte Gram, Cristine Betzer, Clara Perez-Gozalbo, Marjo Beltoja, Madhu Nagaraj, Jie Wang, Jan S. Nowak, Mingdong Dong, Daniel Otzen, Ümit Akbey, Sissel Schmidt, Morten Meyer, Marina Romero-Ramos, Benoit Giasson, Poul H. Jensen. Multiple system atrophy-associated oligodendroglial protein p25 alpha stimulates formation of novel alpha-synuclein strain with enhanced neurodegenerative potential. Acta Neuropathologica 142, 1, 87-115, 2021. DOI: 10.1007/s00401-021-02316-0
Madhu Nagaraj; Mumdooh Ahmed; Jeppe Lyngso; Brian S. Vad; Andreas Bogglid; Anne Filipsen; Jan Skov Pederson; Daniel Otzen, Ümit Akbey. Predicted Loop Regions Promote Aggregation: A Study of Amyloidogenic Domains in the Functional Amyloid FapC. Journal of Molecular Biology 432, 7, 2232-2252, 2020. DOI: /10.1016/j.jmb.2020.01.044
Anne Diehl, Yvette Roske , Linda Ball , Anup Chowdhury , Mattias Hiller , Noel Moliere , Regina Kramer , Daniel Stöppler, Catherine L. Worth , Brigitte Schlegel , Martina Leidert , Nils Cremer , Natalja Erdmann , Daniel Lopez , Heike Stephanowitz , Eberhard Krause , Barth-Jan van Rossum , Peter Schmieder , Udo Heinemann , Kürşad Turgay , Ümit Akbey , Hartmut Oschkinat. Structural changes of TasA in biofilm formation of Bacillus. PNAS, 27, 115 (13), 3237-3242, 2018. DOI: 10.1073/pnas.1718102115
Sandra Muschiol, Simon Erlendsson, Marie-Stephanie Aschtgen, Vitor Oliveira1, Peter Schmieder, Casper de Lichtenberg, Kaare Teilum, Thomas Boesen, Ümit Akbey, Birgitta Henriques-Normark. Structure of the competence pilus major pilin ComGC in Streptococcus pneumonia. Journal of Biological Chemistry, 292, 14134-14146, 2017. DOI: 10.1074/jbc.M117.787671
Structural biology of native Bacterial Biofilms
We use modern NMR spectroscopy to study native bacterial biofilms. We utilize multidimensional magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native Pseudomonas fluorescens colony biofilms as our model biofilm system at natural abundance without isotope-labelling. Other biofilms closely related to chronic human infections are also in the scope of Akbey Lab. By using a high-resolution INEPT-based 2D 1H-13C ssNMR spectrum and thorough peak deconvolution at the 1D ssNMR spectra, approximately 80/134 (in 1D/2D) distinct biofilm chemical sites were identified. We compared CP and INEPT 13C ssNMR spectra to different signals originating from the mobile and rigid fractions of the biofilm, and qualitatively determined dynamical changes by comparing CP buildup behaviors. Protein and polysaccharide signals were differentiated and identified by utilizing FapC protein signals as a template, a biofilm forming functional amyloid from Pseudomonas. We identified several biofilm polysaccharide species such as glucose, mannan, galactose, heptose, rhamnan, fucose and N-acylated mannuronic acid by using 1H and 13C chemical shifts obtained from the 2D spectrum. To our knowledge, this study marks the first high-resolution multidimensional ssNMR characterization of a native bacterial biofilm. Our experimental pipeline can be readily applied to other in vitro biofilm model systems and natural biofilms and holds the promise of making a substantial impact on biofilm research, fostering new ideas and breakthroughs to aid in the development of strategic approaches to combat infections caused by biofilm-forming bacteria.
This research topic is in close collaboration with Prof. Wook Kim from Duquesne University, Pittsburgh, USA and Dr. Fred Mentink from NHFML Tallahassee, USA.
Chang-Hyeock Byeon, , , , , , Ümit Akbey (2023) Journal of Magnetic Resonance. Tapping into the native Pseudomonas Bacterial Biofilm Structure by High-Resolution Multidimensional Solid-State NMR. Journal of Magnetic Resonance, DOI: 10.1016/j.jmr.2023.107587
Developing advanced solid-state MAS NMR methods for proton detection
In my group we develop novel NMR methods to push the limits of the state of the art MAS solid-state NMR to understand molecular details and mechanisms of insoluble and non-crystalline proteins. Proton detected solid-state NMR has made a remarkable progress in the last decade to become a high-resolution and -sensitivity method due to advances in sample preparation, hardware, novel methods such as proton-detection and hyperpolarization (DNP). These allow studies of not only difficult proteins in-vitro but also in their complex in-vivo environment. We also like to combine ssNMR with other exciting structural biology tools.
Andy J. Nieuwkoop, Trent W. Franks, Khristina Rehbein, Anne Diehl, Ümit Akbey, Frank Engelke, Lyndon Emsley, Guido Pintacuda, Hartmut Oschkinat. Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning. J Biomol NMR. 61 (2), 161-71. 2015. DOI: 10.1007/s10858-015-9904-0
Jozef R. Lewandowski, Jean-Nicolas Dumez, Ümit Akbey, Sascha Lange, Lyndon Emsley, Hartmut Oschkinat. Enhanced resolution and coherence lifetimes in the solid-state NMR spectroscopy of perdeuterated systems under ultrafast magic-angle spinning. Phys. Chem. Comm. 2, 17, 2205-2211, 2011. DOI: 10.1021/jz200844n
Ümit Akbey, Sascha Lange, W. Trent Franks, Rasmus Linser, Kristina Rehbein, Anne Diehl, Barth-Jan van Rossum, Bernd Reif, Hartmut Oschkinat. Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy. Journal of Biomolecular NMR. 46 (1), 67-73, 2010. DOI: 10.1007/s10858-009-9369-0.
Developing advanced solid-state MAS NMR methods for deuterium-based protein dynamics
We work on deuterium (2H) based structure and dynamics studies of solid proteins, to understand the function of biological systems better. Traditionally, proton excitation and detection has been utilized for structural biology applications on deuterated proteins. However, this is not the only possible approach for such systems and deuterium is a readily available novel and exciting reporter nucleus to exploit. Deuterium is an abundant spin reservoir and is a promising alternative to proton to be incorporated into the structure and dynamics characterization protocols in perdeuterated proteins. Recently we utilized 2D 2H–13C MAS NMR to determine deuterium quadrupolar coupling in a semi-quantitative manner. Variable-temperature approach was utilized to monitor the temperature induced dynamics changes of a protein between 100 and 320 K. This kind of NMR spectroscopy only requires a double-resonance MAS NMR probe due to the excitation of deuterium and detection of carbon. However, due to signal overlap, especially at lower temperatures, this approach becomes limited. Instead, a 3D 2H–13C–13C is possible for site-specificity, at the expense of long experimental time due to low sensitivity carbon detection, nevertheless feasible. Most recently, we used a special MAS NMR probe with four individual radio-frequency channels, to be able to characterize the backbone Ca deuterons by amide proton detection.1 We demonstrate the use of proton-detected MAS NMR approach, and incorporated deuterium-spin gymnastics into those experimental pulse sequences on modal systems, and will extend the use of them on FuBAs we are interested in.
This research topic is in close collaboration with Prof. Niels Chr. Nielsen from Aarhus University, Denmark
Ümit Akbey. Site-specific protein backbone deuterium 2Hα quadrupolar patterns by proton-detected quadruple-resonance 3D 2HαcαNH MAS NMR spectroscopy. Journal of Magnetic Resonance, 2023 125, 1010861. DOI: 10.1016/j.ssnmr.2023.101861.
Ümit Akbey. Site-specific Protein Methyl Deuterium Quadrupolar Patterns by Proton detected 3D 2H13C1H MAS NMR Spectroscopy. Journal of Biological NMR, 2022. DOI: 10.1007/s10858-021-00388-4
Ümit Akbey. Dynamics of uniformly labelled solid proteins between 100-300 K: A 2D 2H-13C MAS NMR approach. Journal of Magnetic Resonance, 327, 106974, 2021. DOI: 10.1016/j.jmr.2021.106974
Ümit Akbey, Andrew J. Nieuwkoop, Sebastian Wegner, Anja Voreck, Britta Kunert, Priyanga Bandara, Frank Engelke, Niels Chr. Nielsen, Hartmut Oschkinat. Quadruple-Resonance MAS NMR Spectroscopy of Deuterated Solid Proteins. Chem. Int. Ed. 53, 2438-2442, 2014. DOI: 10.1002/anie.201308927
Ümit Akbey, Hartmut Oschkinat, Barth-Jan van Rossum. Double-Nucleus Enhanced Recoupling for Efficient 13C MAS NMR Correlation Spectroscopy of Perdeuterated Proteins. Am. Chem. Soc. 131 (47), 17054–17055, 2009. DOI: 10.1021/ja907493p.
Developing advanced Hyperpolarized DNP NMR methods for Structural Biology
In addition to the development of novel solid-state NMR methods, we also contribute to the hyperpolarization enhanced NMR spectroscopy, mainly by dynamic nuclear polarization (DNP). DNP has been proven to increase NMR sensitivity by several orders of magnitude, that allows the studies of difficult proteins in their complex in-vivo environment, native cells, or bacteria. By contributing to the development of DNP, we utilize it for studying biofilm forming FuBAs. Efficient use of DNP enhanced NMR is a sample dependent optimization process, and sometimes also requires selective isotope-labelled approaches to get the most information out. Due to the signal broadening at the DNP experimental operation temperatures of ~100 K, the resolution maybe limited and this requires tailored research questions to be asked and focused on.
This research topic is in close collaboration with Dr. Fred Mentink from NHFML Tallahassee, USA.
Anna König, Daniel Schölzel, Boran Uluca, Thibault Viennet, Ümit Akbey, Henrike Heise. Hyperpolarized MAS NMR of unfolded and misfolded proteins. ssNMR, 98, 1-11, 2019. DOI: j.ssnmr.2018.12.003
Ümit Akbey and Hartmut Oschkinat. Structural Biology Applications of Solid State MAS DNP NMR. JMR. DOI: 10.1016/j.jmr.2016.04.003
Christian Bretschneider, Ümit Akbey, Fabien Aussenac, Greg Olsen, Akiva Feintuch,, Hartmut Oschkinat, Lucio Frydman. On The Potential of Dynamic Nuclear Polarization Enhanced Diamonds in Solid‐State and Dissolution 13C NMR Spectroscopy. ChemPhysChem. 2016. DOI:1002/cphc.201600301. Cover Article.
Geiger, Michel-Andreas; Orwick-Rydmark, Marcella; Marker, Katharina; et al. Ümit Akbey, Hartmut Oschkinat. Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures. Phys. Chem. Chem. Phys., 2016,18, 30696-30704. DOI: 10.1039/C6CP06154K
Frederic Mentink-Vigier, Ümit Akbey, Akiva Feintuch, Shimon Vega, Hartmut Oschkinat. Theoretical Aspects of Magic Angle Spinning – Dynamic Nuclear Polarization. 258, 102-120, 2015. DOI: 10.1016/j.jmr.2015.07.001
Ümit Akbey, Trant Franks, Arne Linden, Marcella Orwick Rydman, Sascha Lange, Hartmut Oschkinat. Dynamic Nuclear Polarization Enhanced Solid State NMR of Proteins. Springer, Topics in Current Chemistry. 2013. DOI: 10.1007/128_2013_436