Heterogeneous and rate-dependent streptavidin-biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations

Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information of the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds.Comment: 21 pages, 4 figure

AFMBioMed Conference: Paris, France, August 2011

International audienceThe fourth edition of the AFMBioMed Conference, Paris 2011 was organized by the Institut Curie, INSERM, and the Life Science Division of the CEA (DSV). The conference was held at the Institut Curie, Paris, France on 23–26 August and chaired by Dr Simon Scheuring

Carbohydrate–carbohydrate interaction provides adhesion force and specificity for cellular recognition

© 2004 Bucior et al. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of Cell Biology 165 (2004): 529-537, doi:10.1083/jcb.200309005.The adhesion force and specificity in the first experimental evidence for cell–cell recognition in the animal kingdom were assigned to marine sponge cell surface proteoglycans. However, the question whether the specificity resided in a protein or carbohydrate moiety could not yet be resolved. Here, the strength and species specificity of cell–cell recognition could be assigned to a direct carbohydrate–carbohydrate interaction. Atomic force microscopy measurements revealed equally strong adhesion forces between glycan molecules (190–310 piconewtons) as between proteins in antibody–antigen interactions (244 piconewtons). Quantitative measurements of adhesion forces between glycans from identical species versus glycans from different species confirmed the species specificity of the interaction. Glycan-coated beads aggregated according to their species of origin, i.e., the same way as live sponge cells did. Live cells also demonstrated species selective binding to glycans coated on surfaces. These findings confirm for the first time the existence of relatively strong and species-specific recognition between surface glycans, a process that may have significant implications in cellular recognition.This work was supported by the Friedrich Miescher Institute, branch of the Novartis Research Foundation, the M.E. Müller Foundation, and the Swiss National Research Foundatio

The hierarchical assembly of septins revealed by high-speed AFM

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Jiao, F., Cannon, K. S., Lin, Y. C., Gladfelter, A. S., & Scheuring, S. The hierarchical assembly of septins revealed by high-speed AFM. Nature Communications, 11(1), (2020): 5062, doi:10.1038/s41467-020-18778-x.Septins are GTP-binding proteins involved in diverse cellular processes including division and membrane remodeling. Septins form linear, palindromic heteromeric complexes that can assemble in filaments and higher-order structures. Structural studies revealed various septin architectures, but questions concerning assembly-dynamics and -pathways persist. Here we used high-speed atomic force microscopy (HS-AFM) and kinetic modeling which allowed us to determine that septin filament assembly was a diffusion-driven process, while formation of higher-order structures was complex and involved self-templating. Slightly acidic pH and increased monovalent ion concentrations favor filament-assembly, -alignment and -pairing. Filament-alignment and -pairing further favored diffusion-driven assembly. Pairing is mediated by the septin N-termini face, and may occur symmetrically or staggered, likely important for the formation of higher-order structures of different shapes. Multilayered structures are templated by the morphology of the underlying layers. The septin C-termini face, namely the C-terminal extension of Cdc12, may be involved in membrane binding.We thank J. Thorner for the generous gift of the CTE mutant plasmids. K.S.C. was supported in part by a grant from NIGMS under award T32 GM119999 and A.S.G., F.J. and S.S. were supported by NIH RO1 GM130934

Materials and Textile Architecture Analyses for Mechanical Counter-Pressure Space Suits using Active Materials

Mechanical counter-pressure (MCP) space suits have the potential to improve the mobility of astronauts as they conduct planetary exploration activities. MCP suits differ from traditional gas-pressurized space suits by applying surface pressure to the wearer using tight-fitting materials rather than pressurized gas, and represent a fundamental change in space suit design. However, the underlying technologies required to provide uniform compression in a MCP garment at sufficient pressures for space exploration have not yet been perfected, and donning and doffing a MCP suit remains a significant challenge. This research effort focuses on the novel use of active material technologies to produce a garment with controllable compression capabilities (up to 30 kPa) to address these problems. We provide a comparative study of active materials and textile architectures for MCP applications; concept active material compression textiles to be developed and tested based on these analyses; and preliminary biaxial braid compression garment modeling results.United States. National Aeronautics and Space Administration (OCT Space Technology Research Fellowship Grant NNX11AM62H)MIT-Portugal Progra

Co- and post-translational translocation through the protein-conducting channel:analogous mechanisms at work?

Many proteins are translocated across, or integrated into, membranes. Both functions are fulfilled by the 'translocon/translocase', which contains a membrane-embedded proteinconducting channel (PCC) and associated soluble factors that drive translocation and insertion reactions using nucleotide triphosphates as fuel. This perspective focuses on reinterpreting existing experimental data in light of a recently proposed PCC model comprising a front-to-front dimer of SecY or Sec61 heterotrimeric complexes. In this new framework, we propose (i) a revised model for SRP-SR-mediated docking of the ribosome-nascent polypeptide to the PCC; (ii) that the dynamic interplay between protein substrate, soluble factors and PCC controls the opening and closing of a transmembrane channel across, and/or a lateral gate into, the membrane; and (iii) that co-and post-translational translocation, involving the ribosome and SecA, respectively, not only converge at the PCC but also use analogous mechanisms for coordinating protein translocation

Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity

Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins

Flexibility and size heterogeneity of the LH1 light harvesting complex revealed by atomic force microscopy - Functional significance for bacterial photosynthesis

Previous electron microscopic studies of bacterial RC-LH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the

Cryo-Electron Microscopy of Potassium Channel Membrane Proteins

ICS 2301 Design and Analysis of Algorithms SU