Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules.

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa. Polyethylene glycol and Ficoll polymers of different molecular masses were used in buffer solutions to mimic a crowded environment. The reduction in translational mobility of the biological tracer molecules was analyzed as a function of crowder volume fractions and was generally more pronounced in PEG as compared to Ficoll solutions. For several crowding conditions, we observed a molecular sieving effect, in which the diffusion coefficient of larger tracer molecules is reduced to a larger extent than predicted by the Stokes-Einstein relation. By employing a FRET-based biosensor, we also showed that a multiprotein complex is significantly compacted in the presence of macromolecular crowders. Importantly, with respect to sensor in vivo applications, ligand concentration determining sensors would need a crowding specific calibration in order to deliver correct cytosolic ligand concentration.

Two-Dimensional Folding of Polypeptides into Molecular Nanostructures at Surfaces.

Herein we report the fabrication of molecular nanostructures on surfaces via two-dimensional (2D) folding of the nine amino acid peptide bradykinin. Soft-landing electrospray ion beam deposition in conjunction with high-resolution imaging by scanning tunneling microscopy is used to fabricate and investigate the molecular nanostructures. Subnanometer resolved images evidence the large conformational freedom of the molecules if thermal motion is inhibited and the formation of stable uniform dimers of only one specific conformation when diffusion can take place. Molecular dynamics modeling supported by density functional theory calculations give atomically precise insight into the induced-fit binding scheme when the folded dimer is formed. In the absence of solvent, we find a hierarchy of binding strength from polar to nonpolar, manifested in an inverted polar-nonpolar segregation which suppresses unspecific interactions at the rim of the nanostructure. The demonstrated 2D-folding scheme resembles many key properties of its native 3D counterpart and shows that functional, molecular nanostructures on surfaces fabricated by folding could be just as versatile and specific.

Active conformation control of unfolded proteins by hyperthermal collision with a metal surface.

The physical and chemical properties of macromolecules like proteins are strongly dependent on their conformation. The degrees of freedom of their chemical bonds generate a huge conformational space, of which, however, only a small fraction is accessible in thermal equilibrium. Here we show that soft-landing electrospray ion beam deposition (ES-IBD) of unfolded proteins allows to control their conformation. The dynamics and result of the deposition process can be actively steered by selecting the molecular ion beam's charge state or tuning the incident energy. Using these parameters, protein conformations ranging from fully extended to completely compact can be prepared selectively on a surface, as evidenced on the subnanometer/amino acid resolution level by scanning tunneling microscopy (STM). Supported by molecular dynamics (MD) simulations, our results demonstrate that the final conformation on the surface is reached through a mechanical deformation during the hyperthermal ion surface collision. Our experimental results independently confirm the findings of ion mobility spectrometry (IMS) studies of protein gas phase conformations. Moreover, we establish a new route for the processing of macromolecular materials, with the potential to reach conformations that would be inaccessible otherwise.