Day 2 :
Florida State Univ. & National High Magnetic Field Lab, USA
Timothy Cross has more that 30 years of experience characterizing membrane proteins in lipid bilayer environments using solid state NMR of liquid crystalline bilayer preparations of peptides and proteins. This has brought to light a fundamental understanding of membrane protein biophysics that has led to detailed functional characterizations of membrane channels – in particular the gramicidin monovalent cation channel and the influenza A M2 proton channel. In both systems the unique features of the membrane environment play crucial roles in the functional mechanisms and kinetics of ion conductance. Is this understanding of the influence of membrane and lipid environments through the use of solid state NMR that has driven his research at the frontier of membrane protein biophysics.
Statement of the Problem: Unlike water soluble proteins that have a relatively homogeneous environment, membrane proteins exist in a dramatically heterogeneous environment. The result is protein structure that is stabilized by a different balance of molecular interactions for the membrane embedded portion of the protein compared to the water soluble or membrane interfacial regions of the protein. The result is a need to model the membrane environment as closely as possible to that of the native environment for structural, dynamic and functional characterizations. Methodology: Biological solid state NMR provides a unique opportunity to model the membrane environment with liquid crystalline lipid bilayers and a wide variety of lipids. The samples can be prepared either as liposomes for magic angle sample spinning (MAS) or as uniformly oriented samples (OS) for the spectroscopy. The former provides solution like spectra for both distance and isotropic chemical shift restraints, while oriented samples provide absolute restraints that restrain the atomic sites in the protein structure to the bilayer normal. In addition to structural restraints it is possible to characterize the protein’s dynamics and kinetic rates. Findings, Conclusion & Significance: The structure, dynamics and kinetics associated with the M2 proton channel from influenza A have been characterized yielding a unique mechanism for proton transport by this important drug target. In addition, the cholesterol binding to M2 has been found to stabilize the amphipathic helix in the lipid interface an essential feature for this protein’s functional role in viral budding. Recent structural studies of the CrgA protein from Mycobacterium tuberculosis have characterized a dimeric structure stabilized primarily by intermolecular b-sheet in the membrane interfacial region. The protein is part of the cell division apparatus and appears to play a role in recruiting multiple proteins to the divisome, potentially through its transmembrane domain.
1. Zhou, H.-X. & Cross, T.A. (2013) “Influences of Membrane Mimetic Environments on Membrane Protein Structures” Annual Reviews of Biophysics 42:361-392.
2. Sharma, M., Yi, M., Dong, H., Qin, H., Petersen, E., Busath, D.D., Zhou, H.-X. & Cross, T.A. (2010) “Insight into the Mechanism of the Influenza A Proton Channel from a Structure in a Lipid Bilayer” Science 330:509-512.
3. Murray, D., Das, N., Cross, T.A. (2013) “Solid State NMR Strategy for Characterizing Native Membrane Protein Structures” Accounts of Chemical Research 46:2172-2181.
4. Miao, Y., Fu, R., Zhou, H.-X. & Cross, T.A. (2015) Dynamic, Short Hydrogen Bonds in Histidine Tetrad of Full-Length M2 H+ Channel Reveals Tetrameric Structural Heterogeneity and Functional Mechanism” Structure 23:2300-2308.
5. Das, N., Dai, J., Hung, I., Rajagopalan, M., Zhou, H.-X. & Cross, T.A. (2015) “Structure of CrgA, a Cell Division Structural and Regulatory Protein from Mycobacterium tuberculosis in Lipid Bilayers” Proc. Natl. Acad. Sci. 112:E119-E126.
University of Virginia, USA
Wladek Minor is a Harrison Distinguished Professor of Molecular Physiology and Biological Physics at University of Virginia. He is an expert in structural biology and data mining. He is an author of over 190 papers that attracted over 37,000 of citations. His Relative Citations Ratio is above 560.He trained over 80 scientists that currently pursue career in academia, government and industry.
Experimental reproducibility is the cornerstone of scientific research, upon which all progress rests. The veracity of scientific publications is crucial because subsequent lines of investigation rely on previous knowledge. Several recent systematic surveys of academic results published in biomedical journals reveal that a large fraction of representative sets of studies in a variety of fields cannot be reproduced in another laboratory. Big Data approach and especially NIH Big Data to Knowledge (BD2K) program is coming to the rescue.
The goal of the presented research is to provide the biomedical community with a strategy to increase the reproducibility of reported results for a wide range of experiments by building a set of “best practices”, culled by extensive data harvesting and curation combined with experimental verification of the parameters crucial for reproducibility. Experimental verification assisted by the automatic/semi-automatic harvesting of data from laboratory equipment into the already developed sophisticated laboratory information management system (LIMS) will be presented. This data in, information out paradigm will be discussed.
- Zheng H, Cooper DR, Porebski PJ, Shabalin IG, Handing KB, Minor W (2017) CheckMyMetal: a macromolecular metal-binding validation tool. Acta Cryst. D 73: 223-233
- Grabowski M, Minor W (2017) Sharing Big Data. IUCrJ 4: 3-4
- Rupp B, Wlodawer A, Minor W, Helliwell JR, Jaskolski M (2016) Correcting the record of structural publications requires joint effort of the community and journal editors. FEBS J. 283: 4452-4457
- Grabowski M, Langner KM, Cymborowski M, Porebski PJ, Sroka P, Zheng H, Cooper DR, Zimmerman MD, Elsliger MA, Burley SK, Minor W (2016) A public database of macromolecular diffraction experiments. Acta Cryst D 72: 1181-1193
- Niedzialkowska E, Gasiorowska O, Handing KB, Majorek KA, Porebski PJ, Shabalin IG, Zasadzinska E, Cymborowski M, Minor W (2016) Protein purification and crystallization artifacts: The tale usually not told. Protein Science 25: 720-33
University of Nebraska Medical Center, USA
Yuri L. Lyubchenko is Professor of Pharmaceutical Sciences at the University of Nebraska Medical Center, Omaha, NE, USA. His research focuses on understanding fundamental mechanisms underlying health and disease, which are key to developing new and more effective diagnostics and medications. This primarily basic research allows him not only identify new drug targets for small molecule drugs, it also leads to development of the nanotools and methods to discover novel approaches for diagnostic, treatment and disease prevention and to more rapidly determine their efficacy at the molecular level.
The amyloid cascade hypothesis is currently considered as the main model for a vast number of neurodegenerative diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Numerous studies have shown that amyloidogenic proteins are capable of spontaneous assembly into aggregates, and eventually form fibrillar structures found in amyloid or amyloid‐like deposits. However, there is a serious complication with translating current knowledge on amyloid aggregation in vitro to understand the aggregation process in vivo. If the critical concentration for the spontaneous aggregation of Aβ peptide in vitro is in the micromolar range, physiological concentrations of Aβ are in the low nanomolar range making impossible amyloids to assemble. We have discovered a novel on-surface aggregation pathway that allows for spontaneous assembly of amyloid beta peptides at the physiological concentration range. Our combined experimental and computer modeling approaches demonstrate that the on-surface aggregation is a dynamic process, so the assembled aggregate can dissociate from the surface to the bulk solution. As a result, the dissociated oligomers can play roles of seeds for aggregation in the bulk solution, or start a neurotoxic effect such as phosphorylation of the tau protein to initiate its misfolding and aggregation. Both processes lead to neurodegeneration. Importantly, in the vast majority of cases, we found that aggregates formed on the surface are oligomers, which are considered to be the most neurotoxic amyloid aggregates. Therefore, we posit that on-surface aggregation is the mechanism by which neurotoxic amyloid aggregates are produced under physiological conditions. A change in membrane properties leading to an increase in affinity of amyloid proteins to the membrane surface facilitates the assembly of stable oligomers. The proposed model is a significant departure from the current model as it directs the development of treatments and preventions towards approaches that control the cell membranes properties and composition preventing the on-surface aggregation process.
1. Lyubchenko, Y. L. (2014) Centromere chromatin: a loose grip on the nucleosome?, Nat Struct Mol Biol 21, 8.
2. Lyubchenko, Y. L., and Shlyakhtenko, L. S. (2015) Chromatin imaging with time-lapse atomic force microscopy, Methods Mol Biol 1288, 27-42.
3. Lyubchenko, Y. L., and Shlyakhtenko, L. S. (2015) Atomic Force Microscopy Imaging and Probing of Amyloid Nanoaggregates, In Handbook of Clinical Nanomedicine: From Bench to Bedside (Bawa, R., Audette, G. & Rubinstein, I., Ed.), p 1500. , Pan Stanford Publishing, Singapore. .
4. Sun, Z., Tan, H. Y., Bianco, P. R., and Lyubchenko, Y. L. (2015) Remodeling of RecG Helicase at the DNA Replication Fork by SSB Protein, Sci Rep 5, 9625.
5. Proctor, E. A., Fee, L., Tao, Y., Redler, R. L., Fay, J. M., Zhang, Y., Lv, Z., Mercer, I. P., Deshmukh, M., Lyubchenko, Y. L., and Dokholyan, N. V. (2016) Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis, Proc Natl Acad Sci U S A 113, 614-619.