We investigate the kinetic pathways of folding and unfolding of the designed miniprotein Trp-cage in explicit solvent. Straightforward molecular dynamics and replica exchange methods both have severe convergence problems, whereas transition path sampling allows us to sample unbiased dynamical pathways between folded and unfolded states and leads to deeper understanding of the mechanisms of (un)folding. In contrast to previous predictions employing an implicit solvent, we find that Trp-cage folds primarily (80% of the paths) via a pathway forming the tertiary contacts and the salt bridge, before helix formation. The remaining 20% of the paths occur in the opposite order, by first forming the helix. The transition states of the rate-limiting steps are solvated native-like structures. Water expulsion is found to be the last step upon folding for each route. Committor analysis suggests that the dynamics of the solvent is not part of the reaction coordinate. Nevertheless, during the transition, specific water molecules are strongly bound and can play a structural role in the folding.protein folding ͉ reaction coordinate ͉ transition path sampling ͉ replica exchange ͉ transition state ensemble E lucidating the mechanism by which proteins fold into their native state remains a central issue in molecular biology. For single domain two-state folding proteins, several decades of experimental, theoretical, and simulation studies have revealed two major qualitative folding mechanisms. In the diffusion-collision mechanism (1), proteins first form secondary structure elements followed by a diffusive search toward the tertiary native state structure. In the nucleation-condensation mechanism (2), a nucleus of crucial tertiary contacts is made, around which the native structure condensates. In recent years, these two mechanisms were combined in a unified view (3).By bridging the gap between experiments and computer simulation, the discovery of small and fast folding proteins has contributed much to the understanding of generic folding mechanisms. The fastest of those is the designed 20-residue miniprotein Trp-cage (NLYIQ WLKDG GPSSG RPPPS) (4), which folds in 4 s to a native state with an ␣-helix, a salt bridge, and a polyproline II helix shielding the central tryptophan from solvent. Laser temperaturejump spectroscopy experiments by Qiu et al. (5) indicated two-state folding. Subsequently, fluorescent correlation spectroscopy by Neuweiler et al. (6) revealed that the protein (un)folds in a more complicated manner via an intermediate molten globule-like state, characterized by exposure of the tryptophan to the solvent. It remains unclear at what stage of folding the helix is being formed. Recent UV-resonance Raman spectroscopy measurements show some evidence of a helical structure in the denaturated state of Trp-cage, and thus suggest that an early formation of the helix is possible (7). Many molecular dynamics (MD) simulations were performed to investigate thermodynamic stability of the protein and elucidate possible folding p...

We report rate constant calculations and a reaction coordinate analysis of the rate-limiting folding and unfolding process of the Trp-cage mini-protein in explicit solvent using transition interface sampling. Previous transition path sampling simulations revealed that in this (un)folding process the protein maintains its compact configuration, while a (de)increase of secondary structure is observed. The calculated folding rate agrees reasonably with experiment, while the unfolding rate is 10 times higher. We discuss possible origins for this mismatch. We recomputed the rates with the forward flux sampling method, and found a discrepancy of four orders of magnitude, probably caused by the method's higher sensitivity to the choice of order parameter with respect to transition interface sampling. Finally, we used the previously computed transition path-sampling ensemble to screen combinations of many order parameters for the best model of the reaction coordinate by employing likelihood maximization. We found that a combination of the root mean-square deviation of the helix and of the entire protein was, of the set of tried order parameters, the one that best describes the reaction coordination.

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.antibody recognition | X-ray crystallography | electron microscopy

Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.