Significance Hydrogen bonding is one of the characteristic features of macromolecules. Therefore, it is not surprising that extensive research has gone into understanding the energetics and importance of hydrogen bonding. All of these studies focused on hydrogen bonds that involve a single donor and acceptor pair. Yet the H-bonding potential of many acceptors leads to the phenomenon of overcoordination between two donors and one acceptor. Here we have used both experimental approaches and computational analyses to measure the strength of such bifurcated hydrogen bonds and show that their energy is between 60% and 50% of canonical H bonds. Finally, we show how the energetics of bifurcated H bonds directly impact amino acid side-chain structure.

Like-charged solid interfaces repel and separate from one another as much as possible. Charged interfaces composed of self-assembled charged-molecules such as lipids or proteins are ubiquitous. The present study shows that although charged lipid-membranes are sufficiently rigid, in order to swell as much as possible, they deviate markedly from the behavior of typical like-charged solids when diluted below a critical concentration (ca. 15 wt %). Unexpectedly, they swell into lamellar structures with spacing that is up to four times shorter than the layers should assume (if filling the entire available space). This process is reversible with respect to changing the lipid concentration. Additionally, the research shows that, although the repulsion between charged interfaces increases with temperature, like-charged membranes, remarkably, condense with increasing temperature. This effect is also shown to be reversible. Our findings hold for a wide range of conditions including varying membrane charge density, bending rigidity, salt concentration, and conditions of typical living systems. We attribute the limited swelling and condensation of the net repulsive interfaces to their self-assembled character. Unlike solids, membranes can rearrange to gain an effective entropic attraction, which increases with temperature and compensates for the work required for condensing the bilayers. Our findings provide new insight into the thermodynamics and self-organization of like-charged interfaces composed of self-assembled molecules such as charged biomaterials and supramolecular assemblies that are widely found in synthetic and natural constructs.

The polarity pattern of a macromolecule is of utmost importance to its structure and function. For example, one of the main driving forces for protein folding is the burial of hydrophobic residues. Yet polarity remains a difficult property to measure experimentally, due in part to its nonuniformity in the protein interior. Herein, we show that Fourier transform infrared (FTIR) linewidth analysis of noninvasive 1-13C18O labels can be used to obtain a reliable measure of the local polarity, even in a highly multiphasic system, such as a membrane protein. We show that in the Influenza M2 H+ channel, residues that line the pore are located in an environment that is as polar as fully solvated residues, while residues that face the lipid acyl chains are located in an apolar environment. Taken together, FTIR linewidth analysis is a powerful, yet chemically nonperturbing approach to examine one of the most important properties in proteins: polarity.