Forces guiding assembly of light-harvesting complex 2 in native membranes

Abstract

Interaction forces of membrane protein subunits are of importance in their structure, assembly, membrane insertion, and function. In biological membranes, and in the photosynthetic apparatus as a paradigm, membrane proteins fulfill their function by ensemble actions integrating a tight assembly of several proteins. In the bacterial photosynthetic apparatus light-harvesting complexes 2 (LH2) transfer light energy to neighboring tightly associated core complexes, constituted of light-harvesting complexes 1 (LH1) and reaction centers (RC). While the architecture of the photosynthetic unit has been described, the forces and energies assuring the structural and functional integrity of LH2, the assembly of LH2 complexes, and how LH2 interact with the other proteins in the supramolecular architecture are still unknown. Here we investigate the molecular forces of the bacterial LH2 within the native photosynthetic membrane using atomic force microscopy single-molecule imaging and force measurement in combination. The binding between LH2 subunits is fairly weak, of the order of k B T , indicating the importance of LH2 ring architecture. In contrast LH2 subunits are solid with a free energy difference of 90 k B T between folded and unfolded states. Subunit α-helices unfold either in one-step, α-and β-polypeptides unfold together, or sequentially. The unfolding force of transmembrane helices is approximately 150 pN. In the two-step unfolding process, the β-polypeptide is stabilized by the molecular environment in the membrane. Hence, intermolecular forces influence the structural and functional integrity of LH2.atomic force microscopy | photosynthesis | protein unfolding | membrane protein assembly M embrane protein function is intimately linked and influenced by its structural integrity and molecular environment (1-3). Photosynthesis is a paradigm of such orchestrated molecular action, for which the implicated proteins have evolved to form supramolecular assemblies (4). Knowledge of supramolecular architecture and interaction forces and energies are indispensable for a complete understanding of a membrane protein structure and function.The atomic force microscope (AFM) (5) can be used as a high-resolution imaging (6) and as a force-measurement tool (7). Most elegantly the two applications were combined allowing the attribution of structural changes to force events (8). More recently, the AFM has proven the unique tool for studying the supramolecular assembly of the bacterial photosynthetic unit (9-12) and has provided a solid basis for the understanding of the ensemble function of the photosynthetic proteins (4, 13-15).The light-harvesting process in photosynthetic bacteria starts with photon capture at the peripheral light-harvesting complex 2 (LH2) that transfers the absorbed excitation energy to the light-harvesting complex 1 (LH1) associated with the reaction center (RC). The LH2 structure is well described by X-ray crystallography (16, 17) and AFM in native membranes (12,(18)(19)(20). Typically, the LH2 complexes appe...

Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states

“…We assign this mechanism for two other major reasons: (i) We can eliminate other states of LH2 (triplets, (19) and cations recombine on a picosecond timescale (26). (ii) Each photon deposits sufficient energy into the PPC (∼50-100 kT) to easily move it out of the ground conformational state (27). Furthermore, the reverse (k B→A ) rate of hundreds of milliseconds suggests that state B is a metastable conformation that returns to the ground conformation via thermal fluctuations, as shown by the intensity independence of k B→A .…”

Phycobilisome Mobility and Its Role in the Regulation of Light Harvesting in Red Algae

“…Therefore, macromolecular crowding of proteins has been used to rationalize the restricted protein mobility in thylakoid membranes of chloroplasts (Kirchhoff, 2008a(Kirchhoff, , 2008b. Indeed, atomic force microscopy studies have shown that there is a dense packing and interaction of complexes in the photosynthetic membranes (Liu et al, 2011). Therefore, the diffusion of photosynthetic proteins in the thylakoid membrane is rather slow, and it increases only in less crowded parts of thylakoids (Kirchhoff et al, 2013).…”

Self-Assembly Stability and Variability of Bacterial Microcompartment Shell Proteins in Response to the Environmental Change

“…The BMC shell, consisting of numerous protein homologs, is an ideal system for studying protein self-assembly and interactions. As a powerful technique for analyzing biomembrane organization, protein assembly, and physical interactions that are highly relevant to the physiological roles of biological systems [32,35,38,39], AFM has been exploited to visualize the organization and self-assembly dynamics of BMC shell proteins and the architectures and mechanical features of BMC structures [12,30,31,[40][41][42]. This work represents, to our knowledge, the first quantitative determination of the self-assembly dynamics of BMC shell proteins in the formation of two-dimensional sheets in response to environmental changes using AFM.…”

“…HS-AFM images were captured at 30 or 40 Hz in solution in AC mode using a JPK NanoWizard ULTRA speed AFM equipped with an ULTRA Speed 2.8 μm scanner and Ultra-Short Cantilever USC-0.3 MHz probes (NanoWorld). Minimal loading forces of~100 picoNewton were applied during AFM imaging to reduce disturbance of protein assembly [12,[32][33][34][35][36].…”