https://www.zora.uzh.ch/id/eprint/44154/1/Rabe1.pdf

Understanding protein adsorption phenomena at solid surfaces

The formation of crystalline nanoparticles starts with nucleation and control of nucleation is crucial for the control of the number, size, perfection, polymorph modification and other characteristics of particles. Recently, there have been significant advances in the understanding of the mechanism of nucleation of crystals in solution. The most significant of these is the two-step mechanism of nucleation, according to which the crystalline nucleus appears inside pre-existing metastable clusters of size several hundred nanometers, which consist of dense liquid and are suspended in the solution. While initially proposed for protein crystals, the applicability of this mechanism has been demonstrated for small-molecule organic and inorganic materials, colloids, and biominerals. This mechanism helps to explain several long-standing puzzles of crystal nucleation in solution: nucleation rates which are many orders of magnitude lower than theoretical predictions, nucleation kinetic dependencies with steady or receding parts at increasing supersaturation, the role of heterogeneous substrates for polymorph selection, the significance of the dense protein liquid, and others. More importantly, this mechanism provides powerful tools for control of the nucleation process by varying the solution thermodynamic parameters so that the volume occupied by the dense liquid shrinks or expands.

The two-step mechanism of nucleation of crystals in solution.

Antifreeze Proteins: Structures and Mechanisms of Function.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.

/pdf/structural-studies-of-the-streptavidin-binding-loop-2c9ydplq1r.pdf

Structural studies of the streptavidin binding loop.

Thermal hysteresis proteins.

The Mechanism of Protein Crystal Growth from Lipid Layers

Single crystals of a type III antifreeze polypeptide from ocean pout.

Synthetic peptides were used as models for studying the conformation of ice nucleation proteins. We chemically synthesized four peptides (16-, 24-, 32-, and 48-mer) that consisted of two to six repeats of the consensus repeating octapeptide unit of ice nucleation proteins and evaluated their conformation by circular dichroism spectroscopy. These model peptides exist predominantly as random coils in aqueous solution, but adopt alpha-helical structures in the presence of trifluoroethanol. The stability of their secondary structures was investigated by monitoring the pH and time dependence of their circular dichroism spectra. Our results indicated that the alpha-helical content of the 48-mer exhibited a significant pH dependence, while that of the 24- and 32-mer peptides did not. The 32-mer was the only peptide that transformed from the alpha-helical to a beta-sheet structure upon storage. We suggest that the overall conformation of the ice nucleation protein could be a beta-sheet.

Paul Ala

Papers

The Mechanism of Protein Crystal Growth from Lipid Layers

Single crystals of a type III antifreeze polypeptide from ocean pout.

Synthesis and characterization of a fragment of an ice nucleation protein.

Crystallization of Bacillus subtilis tryptophanyl-tRNA synthetase