Ergodic and nonergodic processes coexist in the plasma membrane as observed by single-molecule tracking

Aubrey V. Weigel, Blair Simon, Michael M. Tamkun, and Diego Krapf

Diffusion in the plasma membrane of living cells is often found to display anomalous dynamics. However, the mechanism underlying this diffusion pattern remains highly controversial. Here, we study the physical mechanism underlying Kv2.1 potassium channel anomalous dynamics using single-molecule tracking. Our analysis includes both time series of individual trajectories and ensemble averages. We show that an ergodic and a nonergodic process coexist in the plasma membrane. The ergodic process resembles a fractal structure with its origin in macromolecular crowding in the cell membrane. The nonergodic process is found to be regulated by transient binding to the actin cytoskeleton and can be accurately modeled by a continuous-time random walk. When the cell is treated with drugs that inhibit actin polymerization, the diffusion pattern of Kv2.1 channels recovers ergodicity. However, the fractal structure that induces anomalous diffusion remains unaltered. These results have direct implications on the regulation of membrane receptor trafficking and signaling.

DOI

Journal: Proceedings of the National Academy of Sciences

Random-Coil:α-Helix Equilibria as a Reporter for the LewisX–LewisX Interaction

1. Timothy M. Altamore, 
2. Christian Fernández-García, 
3. Andrew H. Gordon, 
4. Tina Hübscher, 
5. Dr. Netnapa Promsawan, 
6. Maxim G. Ryadnov, 
7.  Andrew J. Doig, 
8. Derek N. Woolfson, 
9. Timothy Gallagher

1. Probing weak interactions: A peptide random-coil:α-helix equilibrium has been used to identify a weak carbohydrate–carbohydrate interaction (CCI). Glucose and lactose destabilized the helical conformer while Lewis^X trisaccharide led to increased helicity. Though small, the trend observed indicates that this peptide reporter can detect a single CCI in isolation.

1. DOI

1. Journal: Angewandte Chemie

Locating the Barrier for Folding of Single Molecules under an External Force

Olga K. Dudko, Thomas G. W. Graham, and Robert B. Best


Single-molecule pulling experiments on the folding of biomolecules are usually interpreted with one-dimensional models in which the dynamics occurs on the “pulling coordinate.” Paradoxically, the free-energy profile along this coordinate may lack a refolding barrier, yet a barrier is known to exist for folding; thus, it has been argued that pulling experiments do not probe folding. Here, we show that transitions monitored in pulling experiments probe the true folding barrier but that the barrier may be hidden in the projection onto the pulling coordinate. However, one-dimensional theory using the pulling coordinate still yields physically meaningful energy landscape parameters.


DOI


Journal: Physical Review Letters

GB1 Is Not a Two-State Folder: Identification and Characterization of an On-Pathway Intermediate

Angela Morrone, Rajanish Giri, Rudesh D. Toofanny, Carlo Travaglini-Allocatelli, Maurizio Brunori, Valerie Daggett, and Stefano Gianni

The folding pathway of the small α/β protein GB1 has been extensively studied during the past two decades using both theoretical and experimental approaches. These studies provided a consensus view that the protein folds in a two-state manner. Here, we reassessed the folding of GB1, both by experiments and simulations, and detected the presence of an on-pathway intermediate. This intermediate has eluded earlier experimental characterization and is distinct from the collapsed state previously identified using ultrarapid mixing. Failure to identify the presence of an intermediate affects some of the conclusions that have been drawn for GB1, a popular model for protein folding studies.


DOI


Journal: Biophysical Journal

Probing ribosomal protein–RNA interactions with an external force

Pierre Mangeol, Thierry Bizebard, Claude Chiaruttini, Marc Dreyfus, Mathias Springer, and  Ulrich Bockelmann


Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.


DOI


Journal: Proceedings of the National Academy of Sciences 

Thermodynamic efficiency and mechanochemical coupling of F1-ATPase

1. Shoichi Toyabe, 
2. Takahiro Watanabe-Nakayama, 
3. Tetsuaki Okamoto,
4. Seishi Kudo, and 
5. Eiro Muneyuki

1. F₁-ATPase is a nanosized biological energy transducer working as part of F_oF₁-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F₁-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F₁-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of F_oF₁-ATP synthase. We found that the maximum work performed by F₁-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F₁-ATPase.

1. DOI

1. Journal: 
Proceedings of the National Academy of Sciences

Dynamics of protein folding and cofactor binding monitored by single-molecule force spectroscopy

Yi Cao and Hongbin Li


Many proteins in living cells require cofactors to carry out their biological functions. To reach their functional states, these proteins need to fold into their unique three-dimensional structures in the presence of their cofactors. Two processes, folding of the protein and binding of cofactors, intermingle with each other, making the direct elucidation of the folding mechanism of proteins in the presence of cofactors challenging. Here we use single-molecule atomic force microscopy to directly monitor the folding and cofactor binding dynamics of an engineered metal-binding protein G6-53 at the single-molecule level. Using the mechanical stability of different conformers of G6-53 as sensitive probes, we directly identified different G6-53 conformers (unfolded, apo- and Ni²⁺-bound) populated along the folding pathway of G6-53 in the presence of its cofactor Ni²⁺. By carrying out single-molecule atomic force microscopy refolding experiments, we monitored kinetic evolution processes of these different conformers. Our results suggested that the majority of G6-53 folds through a binding-after-folding mechanism, whereas a small fraction follows a binding-before-folding pathway. Our study opens an avenue to utilizing force spectroscopy techniques to probe the folding dynamics of proteins in the presence of cofactors at the single-molecule level, and we anticipated that this method can be used to study a wide variety of proteins requiring cofactors for their function.


DOI


Journal: Biophysical Journal