Strings and stripes formed by a protein system interacting via a single-patch attraction.

The phase behavior of lactoferrin has been studied as a function of concentration at a pH and ionic strength where lactoferrin is known to interact effectively via a patch-patch attraction. In contrast to isotropic attractive potentials, the directional attraction gives rise to a different phase or solution behavior. At low concentrations, the protein dimerizes. As the concentration is increased, the protein self-assembles into elongated, stripe-like structures at intermediate protein concentrations, a behavior which has been predicted for the case of attractive one-patch colloids. The stripe phase is surprisingly difficult to detect using conventional techniques, i.e. small-angle X-ray scattering, since only a small fraction of the proteins participate in the stripes combined with sedimentation due to micron-sized entities. This is circumvented by monitoring the change in the overall protein concentration by static light scattering and the stripe formation can be followed. For visualization of the structures cryo-TEM is used.

Investigation at residue level of the early steps during the assembly of two proteins into supramolecular objects.

Understanding the driving forces governing protein assembly requires the characterization of interactions at molecular level. We focus on two homologous oppositely charged proteins, lysozyme and α-lactalbumin, which can assemble into microspheres. The assembly early steps were characterized through the identification of interacting surfaces monitored at residue level by NMR chemical shift perturbations by titrating one (15)N-labeled protein with its unlabeled partner. While α-lactalbumin has a narrow interacting site, lysozyme has interacting sites scattered on a broad surface. The further assembly of these rather unspecific heterodimers into tetramers leads to the establishment of well-defined interaction sites. Within the tetramers, most of the electrostatic charge patches on the protein surfaces are shielded. Then, hydrophobic interactions, which are possible because α-lactalbumin is in a partially folded state, become preponderant, leading to the formation of larger oligomers. This approach will be particularly useful for rationalizing the design of protein assemblies as nanoscale devices.

Enhanced protein steering: cooperative electrostatic and van der Waals forces in antigen-antibody complexes.

We study the association of the cationic protein lysozyme with several almost neutral protein fragments but with highly uneven charge distributions. Using mesoscopic protein models, we show how electrostatic interactions can align or steer protein complexes into specific constellations dictated by the specific charge distributions of the interacting biomolecules. Including van der Waals forces significantly amplifies the electrostatically induced orientational steering at physiological solution conditions, demonstrating that different intermolecular interactions can work in a cooperative way in order to optimize specific biochemical mechanisms. Individually, the electrostatic and van der Waals interactions lead only to a relatively weak intermolecular alignment, but when combined, the effect increases significantly.

Association and electrostatic steering of alpha-lactalbumin-lysozyme heterodimers.

The salt and pH dependent association of hen egg white lysozyme with alpha-lactalbumin whey proteins has been studied using molecular level Monte Carlo simulations. A highly uneven charge distribution of alpha-lactalbumin leads to strongly ordered heterodimers that may facilitate the formation of structured, mesoscopic aggregates. This electrostatic steering gives rise to 80% alignment at 5 mM 1 : 1 salt which, due to screening, diminishes to 60% at 100 mM salt. The free energy of interaction minima, dominated by electrostatics, ranges between -9 kT at 1 mM salt to -2 kT at 100 mM (neutral pH). Calculated osmotic second virial cross coefficients indicate complexation in the pH interval 6-10. Multivalent ions are found to effectively destabilize the protein complex and, at constant ionic strength, the order is La(3+) > Ca(2+) > Mg(2+) > Na(+). Upon binding of calcium to alpha-lactalbumin both the interaction and orientational alignment with lysozyme are reduced due to induced changes in the whey protein charge distribution. This potentially explains the experimentally observed absence of supramolecular structuring for the calcium loaded holo alpha-lactalbumin. Where available, good agreement is found with experimental data.

Concentration-Induced Association in a Protein System Caused by a Highly Directional Patch Attraction.

Self-association of the protein lactoferrin is studied in solution using small-angle X-ray scattering techniques. Effective static structure factors have been shown to exhibit either a monotonic or a nonmonotonic dependence on protein concentration in the small wavevector limit, depending on salt concentration. The behavior correlates with a nonmonotonic dependence of the second virial coefficient on salt concentration, such that a maximum appears in the structure factor at a low protein concentration when the second virial coefficient is negative and close to a minimum. The results are interpreted in terms of an integral equation theory with explicit dimers, formulated by Wertheim, which provides a consistent framework able to explain the behavior in terms of a monomer-dimer equilibrium that appears because of a highly directional patch attraction. Short attraction ranges preclude trimer formation, which explains why the protein system behaves as if it were subject to a concentration-dependent isotropic protein-protein attraction. Superimposing an isotropic interaction, comprising screened Coulomb repulsion and van der Waals attraction, on the patch attraction allows for a semiquantitative modeling of the complete transition pathway from monomers in the dilute limit to monomer-dimer systems at somewhat higher protein concentrations.

Charge-induced patchy attractions between proteins.

Static light scattering (SLS) combined with structure-based Monte Carlo (MC) simulations provide new insights into mechanisms behind anisotropic, attractive protein interactions. A nonmonotonic behavior of the osmotic second virial coefficient as a function of ionic strength is here shown to originate from a few charged amino acids forming an electrostatic attractive patch, highly directional and complementary. Together with Coulombic repulsion, this attractive patch results in two counteracting electrostatic contributions to the interaction free energy which, by operating over different length scales, is manifested in a subtle, salt-induced minimum in the second virial coefficient as observed in both experiment and simulations.

Anisotropic Interactions in Protein Mixtures: Self Assembly and Phase Behavior in Aqueous Solution.

Recent experimental studies show that oppositely charged proteins can self-assemble to form seemingly stable microspheres in aqueous salt solutions. We here use parallel tempering Monte Carlo simulations to study protein phase separation of lysozyme/α-lactalbumin mixtures and show that anisotropic electrostatic interactions are important for driving protein self-assembly. In both dilute and concentrated protein phases, the proteins strongly align according to their charge distribution. While this alignment can be greatly diminished by a single point mutation, phase separation is completely suppressed when neglecting electrostatic anisotropy. The results highlight the importance of subtle electrostatic interactions even in crowded biomolecular environments where other short-ranged forces are often thought to dominate.

Molecular evidence of stereo-specific lactoferrin dimers in solution.

Gathering experimental evidence suggests that bovine as well as human lactoferrin self-associate in aqueous solution. Still, a molecular level explanation is unavailable. Using force field based molecular modeling of the protein-protein interaction free energy we demonstrate (1) that lactoferrin forms highly stereo-specific dimers at neutral pH and (2) that the self-association is driven by a high charge complementarity across the contact surface of the proteins. Our theoretical predictions of dimer formation are verified by electrophoretic mobility and N-terminal sequence analysis on bovine lactoferrin.