Introduction

Macromolecular crystallization in the structural genomics era

S-layer recrystallization is part of, at least, two major scientific and technological challenges: a model system to study 2-D polymer crystal formation and a bottom-up approach to build biomimetic and functional interfaces. In particular, the self-assembly process of the bacterial surface layer protein (SbpA) might be influenced by two different interactions: protein/substrate and protein/protein. In this work, we are taking advantage of the versatility of already well known polymer brushes to study the self-assembly mechanism of the S-protein SbpA. In particular, the recrystallization pathway of the protein has been modified by the underlying polymer charge and accessible volume offered by the positively charged poly{[2-(methacryloyloxy)ethyl] trimethylammonium chloride} (PMETAC), the negative poly(sulfo propyl methacrylate) (PSPM) and the non-charged poly(N -isopropyl acrylamide) (PNIPAAm). The recrystallization pathways on polymer brushes have been compared with the bacterial biomimetic case represented by the secondary cell wall polymer (SCWP). Results concerning protein adsorption kinetics, viscoelastic properties, crystal lattice parameters and domain formation are presented, and have been obtained with Quartz Crystal Microbalance with Dissipation Monitoring (QCMD) and Atomic Force Microscopy (AFM).

Two-dimensional protein crystallization on lipid monolayers at a quiescent air/water interface is now a well-established process, but it only operates under a very restricted set of conditions and on a very slow time scale. We have recently been able to significantly extend the conditions under which the proteins will crystallize as well as speed up the process by subjecting the interface to a shearing flow. Here, we investigate the two-way coupling between a protein-laden film and the bulk flow that provides the interfacial shear. This flow in a stationary open cylinder is driven by the constant rotation of the floor. Using the Boussinesq–Scriven surface model for a Newtonian interface coupled to the Navier–Stokes equations for the bulk flow, we find that the surface shear viscosity of protein-laden films under most conditions is small or negligible. This is the case for films subjected to constant shearing flow, regardless of the duration of the flow. However, when the film is intermittently sheared, significant surface shear viscosity is evident. In these cases, the surface shear viscosity is not uniform across the film.

Annexin A5 is a protein that binds to membranes containing negatively charged phospholipids in a calcium-dependent manner. We previously found that annexin A5 self-assembles into two-dimensional (2D) crystals on supported lipid bilayers (SLBs) formed on mica while a monolayer of disordered trimers is formed on SLBs on silica. Here, we investigated in detail and correlated the adsorption kinetics of annexin A5 on SLBs, supported on silica and on mica, with the protein’s 2D self-assembly behavior. For this study, quartz crystal microbalance with dissipation monitoring and ellipsometry were combined with atomic force microscopy. We find, in agreement with previous studies, that the adsorption behavior is strongly dependent on the concentration of dioleoylphosphatidylserine (DOPS) in the SLB and the calcium concentration in solution. The adsorption kinetics of annexin A5 are similar on silica-SLBs and on mica-SLBs, when taking into account the difference in accessible DOPS between silica-SLBs and mica-SLBs. In contrast, 2D crystals of annexin A5 form readily on mica-SLBs, even at low protein coverage (≤10%), whereas they are not found on silica-SLBs, except in a narrow range close to maximal coverage. These results enable us to construct the phase diagram for the membrane binding and the states of 2D organization of annexin A5. The protein binds to the membrane in two different fractions, one reversible and the other irreversible, at a given calcium concentration. The adsorption is determined by the interaction of protein monomers with the membrane. We propose that the local membrane environment, as defined by the presence of DOPS, DOPC, and calcium ions, controls the adsorption and reversibility of protein binding.

This chapter reviews state of the art techniques in biomacromolecular crystallization physics, with emphasis on some of the unresolved problems. Biomacromolecules are crystallized from aqueous solutions. Their solubility decreases when the precipitant, an organic salt, polyethelene glycol or a buffer that changes pH, is added to the biomacromolecular solution. According to the organic chemistry of small molecules, the macromolecule has properties determined by their three-dimensional (3D) structure. This structure directly follows from the molecule composition. The regular polyhedral morphology associated with the layer growth can be replaced by the skeleton and dendritic growth modes following from the thermodynamic or kinetic roughening transitions. This chapter concludes by discussing the impurity distribution as a factor contributing, directly or indirectly, to the crystal imperfection, and inducing irregular molecular orientations within the lattice and conformational changes within the molecules.