- Email: [email protected]
Elucidation of the Molecular Machinery in Photosynthetic Light Harvesting
Tuesday, March 1, 2016 Newest results on the investigation of the photocycle of the bacterial blue light receptor photoactive yellow protein with time-resolved serial femtosecond crystallography at the Linac Coherent Light Source are presented and discussed (Tenboer et al., 2014). Tenboer, J., Basu, S., Zatsepin, N., Pande, K., Milathianaki, D., Frank, M., Hunter, M., Boutet, S., Williams, G. J., Koglin, J. E., Oberthuer, D., Heymann, M., Kupitz, C., Conrad, C., Coe, J., Roy-Chowdhury, S., Weierstall, U., James, D., Wang, D., Grant, T., Barty, A., Yefanov, O., Scales, J., Gati, C., Seuring, C., Srajer, V., Henning, R., Schwander, P., Fromme, R., Ourmazd, A., Moffat, K., Van Thor, J. J., Spence, J. C., Fromme, P., Chapman, H. N. & Schmidt, M. (2014). Science 346, 1242-1246. 1846-Wkshp Watching Proteins Function with Time-Resolved X-ray Diffraction Philip Anfinrud, Friedrich Schotte, Hyun Sun Cho. NIH, Bethesda, MD, USA. To understand how proteins function, it is crucial to know the time-ordered sequence of structural changes that occur as they execute their designed function. We recently developed on the BioCARS beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in proteins with 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore [1], and geminate ligand-binding dynamics in hemoglobin [2]. Briefly, a picosecond laser pulse photoexcites a protein and triggers a structural change, which is probed with a suitably delayed picosecond X-ray pulse. This ‘‘pump-probe’’ approach recovers time-resolved diffraction ‘‘snapshots’’ whose corresponding electron density maps can be stitched together into a real-time movie of the structural changes that ensue. The mechanistically detailed, near-atomic resolution description of the PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/ computational approaches in protein biophysics [3]. This research was supported in part by the Intramural Research Program of the NIH, NIDDK. References: [1] F. Schotte, H.S. Cho, V.R. Kaila, H. Kamikubo, N. Dashdorj, E.R. Henry, T. Graber, R. Henning, M. Wulff, G. Hummer, P.A. Anfinrud Proc. Natl. Acad. Sci. U.S.A. 109, 19256 (2012). [2] F. Schotte, H. S. Cho, J. Soman, M. Wulff, J. S. Olson and P. A. Anfinrud, Chemical Physics, 422, 98-106 (2013). [3] V.R.I. Kaila, F. Schotte, H.S. Cho, G. Hummer, and P.A. Anfinrud, Nature Chemistry, 6, 258 (2014).
Workshop: Frontiers in Biophysical Instrumentation 1847-Wkshp Studying Cell Dynamics using Quantitative Phase Imaging Gabriel Popescu. ECE, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Most living cells do not absorb or scatter light significantly, i.e. they are essentially transparent, or phase objects. Phase contrast microscopy proposed by Zernike in the 1930’s represents a major advance in intrinsic contrast imaging, as it reveals inner details of transparent structures without staining or tagging. While phase contrast is sensitive to minute optical path-length changes in the cell, down to the nanoscale, the information retrieved is only qualitative. Quantifying cell-induced shifts in the optical path-lengths permits nanometer scale measurements of structures and motions in a non-contact, non-invasive manner. Thus, quantitative phase imaging (QPI) has recently become an active field of study and various experimental approaches have been proposed. Recently, we have developed Spatial Light Interference microscopy (SLIM) as a highly sensitive QPI method. Due to its sub-nanometer pathlength sensitivity, SLIM enables interesting structure and dynamics studies over broad spatial (nanometers-centimeters) and temporal (milliseconds-weeks) scales. I will review our recent results on applying SLIM to basic cell studies, such as intracellular transport and cell growth. I will end with a discussion on new method, inspired from interferometry, for measuring cell-generated forces. 1848-Wkshp Probing Single Individual Proteins Unfold and Refold with 1-ms Resolution: Improved AFM-Based Single Molecule Force Spectroscopy Thomas T. Perkins. JILA, NIST & CU-Boulder, Boulder, CO, USA.
375a
Protein folding is understood as a set of transitions between states. Yet, an oversimplified view of the folding process emerges if briefly populated states remain undetected due to ensemble averaging and/or limited instrumental precision. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard cantilever is 50-1,000 ms, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-ms temporal resolution was achieved using ultrashort (L=9 mm) cantilevers on a custom-built, high-speed AFM. By modifying such cantilevers with a focused ion beam, we optimized them for SMFS rather than tappingmode imaging. To enhance usability and throughput, we detected these cantilevers on a commercial AFM retrofitted with a detection laser featuring a 3-mm spot size. The improved capabilities of the modified cantilevers were demonstrated in two biophysical experiments. First, we unfolded a polyprotein, a popular assay, where these cantilevers maintained a 1-ms response time while eliminating cantilever ringing (Qz0.5). In particular, these cantilever had improved short-term precision by avoiding periods of 30-90 pN (peak-topeak) force modulation exhibited by unmodified ultrashort cantilevers undergoing underdamped motion at ~500 kHz. In the second assay, we unfolded bacteriorhodopsin (bR), a model membrane protein. The resulting forceextension curves show unprecedented detail, increasing the number of intermediates resolved while unfolding a pair of transmembrane helices from 2 to 14. Equilibrium measurements revealed the cooperative folding of a 3-amino-acid structural element, resolved those states in <15-ms, and deduced the transition’s underlying energy landscape. These bR results sharpen the picture of membrane protein folding and, more broadly, the instrumental enhancements demonstrate a new experimental regime: studying the equilibrium folding and unfolding of individual proteins with 1-ms resolution. 1849-Wkshp Elucidation of the Molecular Machinery in Photosynthetic Light Harvesting Gabriela Schlau-Cohen. Massachusetts Institute of Technology, Cambridge, MA, USA. In photosynthetic light harvesting, absorbed energy migrates through a protein network to reach a dedicated location for conversion to chemical energy. In higher plants, this energy flow is efficient, directional, and regulated. The regulatory response involves complex and complicated multi-timescale processes that safely dissipate excess energy, thus protecting the system against deleterious photoproducts. We explore the mechanisms behind this photoprotective process in the major light-harvesting protein from green plants (LHCII). By characterizing the conformational states and dynamics of individual proteins, we identify the extent of energy dissipation in single LHCII proteins and how the extent of dissipation changes in response to pH and carotenoid composition, two components known to play a role in photoprotection. From this information, we explore how individual complexes contribute to the balance between efficiency and adaptability in photosynthetic light harvesting. 1850-Wkshp Live-Cell Optical Microscopy Beyond the Diffraction Limit Joerg Bewersdorf. Cell Biology, Yale School of Medicine, New Haven, CT, USA. Optical Nanoscopy (super-resolution) techniques such as STED and FPALM/ PALM/STORM microscopy utilize either targeted or stochastic switching of fluorescent molecules to achieve ~25 nm spatial resolution - about 10-fold below the diffraction limit. However, their primary application has been focused on fixed samples because of (i) a lack of suitable live-cell compatible labels and (ii) time resolutions often limited to minutes, especially for FPALM/ PALM/STORM. In this talk, I will present recent advances in live-cell nanoscopy using newly developed probes, labeling procedures and instrumentation that have been optimized for live-cell imaging.
Workshop: Computational Methods for Ion Permeation and Selection 1851-Wkshp Continuum Theory of Calcium Channels: Fundamental Insights from Simplified Models Dirk Gillespie. Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL, USA.
Workshop: Frontiers in Biophysical Instrumentation 1847-Wkshp Studying Cell Dynamics using Quantitative Phase Imaging Gabriel Popescu. ECE, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Most living cells do not absorb or scatter light significantly, i.e. they are essentially transparent, or phase objects. Phase contrast microscopy proposed by Zernike in the 1930’s represents a major advance in intrinsic contrast imaging, as it reveals inner details of transparent structures without staining or tagging. While phase contrast is sensitive to minute optical path-length changes in the cell, down to the nanoscale, the information retrieved is only qualitative. Quantifying cell-induced shifts in the optical path-lengths permits nanometer scale measurements of structures and motions in a non-contact, non-invasive manner. Thus, quantitative phase imaging (QPI) has recently become an active field of study and various experimental approaches have been proposed. Recently, we have developed Spatial Light Interference microscopy (SLIM) as a highly sensitive QPI method. Due to its sub-nanometer pathlength sensitivity, SLIM enables interesting structure and dynamics studies over broad spatial (nanometers-centimeters) and temporal (milliseconds-weeks) scales. I will review our recent results on applying SLIM to basic cell studies, such as intracellular transport and cell growth. I will end with a discussion on new method, inspired from interferometry, for measuring cell-generated forces. 1848-Wkshp Probing Single Individual Proteins Unfold and Refold with 1-ms Resolution: Improved AFM-Based Single Molecule Force Spectroscopy Thomas T. Perkins. JILA, NIST & CU-Boulder, Boulder, CO, USA.
375a
Protein folding is understood as a set of transitions between states. Yet, an oversimplified view of the folding process emerges if briefly populated states remain undetected due to ensemble averaging and/or limited instrumental precision. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard cantilever is 50-1,000 ms, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-ms temporal resolution was achieved using ultrashort (L=9 mm) cantilevers on a custom-built, high-speed AFM. By modifying such cantilevers with a focused ion beam, we optimized them for SMFS rather than tappingmode imaging. To enhance usability and throughput, we detected these cantilevers on a commercial AFM retrofitted with a detection laser featuring a 3-mm spot size. The improved capabilities of the modified cantilevers were demonstrated in two biophysical experiments. First, we unfolded a polyprotein, a popular assay, where these cantilevers maintained a 1-ms response time while eliminating cantilever ringing (Qz0.5). In particular, these cantilever had improved short-term precision by avoiding periods of 30-90 pN (peak-topeak) force modulation exhibited by unmodified ultrashort cantilevers undergoing underdamped motion at ~500 kHz. In the second assay, we unfolded bacteriorhodopsin (bR), a model membrane protein. The resulting forceextension curves show unprecedented detail, increasing the number of intermediates resolved while unfolding a pair of transmembrane helices from 2 to 14. Equilibrium measurements revealed the cooperative folding of a 3-amino-acid structural element, resolved those states in <15-ms, and deduced the transition’s underlying energy landscape. These bR results sharpen the picture of membrane protein folding and, more broadly, the instrumental enhancements demonstrate a new experimental regime: studying the equilibrium folding and unfolding of individual proteins with 1-ms resolution. 1849-Wkshp Elucidation of the Molecular Machinery in Photosynthetic Light Harvesting Gabriela Schlau-Cohen. Massachusetts Institute of Technology, Cambridge, MA, USA. In photosynthetic light harvesting, absorbed energy migrates through a protein network to reach a dedicated location for conversion to chemical energy. In higher plants, this energy flow is efficient, directional, and regulated. The regulatory response involves complex and complicated multi-timescale processes that safely dissipate excess energy, thus protecting the system against deleterious photoproducts. We explore the mechanisms behind this photoprotective process in the major light-harvesting protein from green plants (LHCII). By characterizing the conformational states and dynamics of individual proteins, we identify the extent of energy dissipation in single LHCII proteins and how the extent of dissipation changes in response to pH and carotenoid composition, two components known to play a role in photoprotection. From this information, we explore how individual complexes contribute to the balance between efficiency and adaptability in photosynthetic light harvesting. 1850-Wkshp Live-Cell Optical Microscopy Beyond the Diffraction Limit Joerg Bewersdorf. Cell Biology, Yale School of Medicine, New Haven, CT, USA. Optical Nanoscopy (super-resolution) techniques such as STED and FPALM/ PALM/STORM microscopy utilize either targeted or stochastic switching of fluorescent molecules to achieve ~25 nm spatial resolution - about 10-fold below the diffraction limit. However, their primary application has been focused on fixed samples because of (i) a lack of suitable live-cell compatible labels and (ii) time resolutions often limited to minutes, especially for FPALM/ PALM/STORM. In this talk, I will present recent advances in live-cell nanoscopy using newly developed probes, labeling procedures and instrumentation that have been optimized for live-cell imaging.
Workshop: Computational Methods for Ion Permeation and Selection 1851-Wkshp Continuum Theory of Calcium Channels: Fundamental Insights from Simplified Models Dirk Gillespie. Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL, USA.