Imaging conformations of holo- and apo-transferrin on the single-molecule level by low-energy electron holography.

Conformational changes play a key role in the biological function of many proteins, thereby sustaining a multitude of processes essential to life. Thus, the imaging of the conformational space of proteins exhibiting such conformational changes is of great interest. Low-energy electron holography (LEEH) in combination with native electrospray ion beam deposition (ES-IBD) has recently been demonstrated to be capable of exploring the conformational space of conformationally highly variable proteins on the single-molecule level. While the previously studied conformations were induced by changes in environment, it is of relevance to assess the performance of this imaging method when applied to protein conformations inherently tied to a function-related conformational change. We show that LEEH imaging can distinguish different conformations of transferrin, the major iron transport protein in many organisms, by resolving a nanometer-scale cleft in the structure of the iron-free molecule (apo-transferrin) resulting from the conformational change associated with the iron binding/release process. This, along with a statistical analysis of the data, which evidences a degree of flexibility of the molecules, indicates that LEEH is a viable technique for imaging function-related conformational changes in individual proteins.

Phase Reconstruction of Low-Energy Electron Holograms of Individual Proteins.

Low-energy electron holography (LEEH) is one of the few techniques capable of imaging large and complex three-dimensional molecules, such as proteins, on the single-molecule level at subnanometer resolution. During the imaging process, the structural information about the object is recorded both in the amplitude and in the phase of the hologram. In low-energy electron holography imaging of proteins, the object's amplitude distribution, which directly reveals molecular size and shape on the single-molecule level, can be retrieved via a one-step reconstruction process. However, such a one-step reconstruction routine cannot directly recover the phase information encoded in the hologram. In order to extract the full information about the imaged molecules, we thus implemented an iterative phase retrieval algorithm and applied it to experimentally acquired low-energy electron holograms, reconstructing the phase shift induced by the protein along with the amplitude data. We show that phase imaging can map the projected atomic density of the molecule given by the number of atoms in the electron path. This directly implies a correlation between reconstructed phase shift and projected mean inner potential of the molecule, and thus a sensitivity to local changes in potential, an interpretation that is further substantiated by the strong phase signatures induced by localized charges.

Three Reasons Why Aspartic Acid and Glutamic Acid Sequences Have a Surprisingly Different Influence on Mineralization.

Understanding the role of polymers rich in aspartic acid (Asp) and glutamic acid (Glu) is the key to gaining precise control over mineralization processes. Despite their chemical similarity, experiments revealed a surprisingly different influence of Asp and Glu sequences. We conducted molecular dynamics simulations of Asp and Glu peptides in the presence of calcium and chloride ions to elucidate the underlying phenomena. In line with experimental differences, in our simulations, we indeed find strong differences in the way the peptides interact with ions in solution. The investigated Asp pentapeptide tends to pull a lot of ions into its vicinity, and many structures with clusters of calcium and chloride ions on the surface of the peptide can be observed. Under the same conditions, comparatively fewer ions can be found in proximity of the investigated Glu pentapeptide, and the structures are characterized by single calcium ions bound to multiple carboxylate groups. Based on our simulation data, we identified three reasons contributing to these differences, leading to a new level of understanding additive-ion interactions.