S.-T. Yau

Quasi-planar nucleus structure in apoferritin crystallization

First-order phase transitions of matter, such as condensation and crystallization, proceed through the formation and subsequent growth of ‘critical nuclei’ of the new phase. The thermodynamics and kinetics of the formation of these critical nuclei depend on their structure, which is often assumed to be a compact, three-dimensional arrangement of the constituent molecules or atoms. Recent molecular dynamics simulations have predicted compact nucleus structures for matter made up of building blocks with a spherical interaction field, whereas strongly anisotropic, dipolar molecules may form nuclei consisting of single chains of molecules. Here we show, using direct atomic force microscopy observations, that the near-critical-size clusters formed during the crystallization of apoferritin, a quasi-spherical protein, and which are representative of the critical nucleus of this system, consist of planar arrays of one or two monomolecular layers that contain 5–10 rods of up to 7 molecules each. We find that these clusters contain between 20 and 50 molecules each, and that the arrangement of the constituent molecules is identical to that found in apoferritin crystals. We anticipate that similarly unexpected critical nucleus structures may be quite common, particularly with anisotropic molecules, suggesting that advanced nucleation theories should treat the critical nucleus structure as a variable.

Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes.

We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.

Solvent entropy contribution to the free energy of protein crystallization.

We show with three proteins that trapping and release of the water molecules upon crystallization is a determinant of the crystallization thermodynamics. With HbC, a strong retrograde solubility dependence on temperature yields a high positive enthalpy of 155 kJ mol(-1), i.e., crystallization is only possible because of the huge entropy gain of 610 J mol(-1) x K(-1), stemming from the release of up to 10 water molecules per protein intermolecular contact. With apoferritin, the enthalpy of crystallization is close to zero. The main component in the crystallization driving force is the entropy gain due to the release upon crystallization of two water molecules bound to one protein molecules in solution. With both proteins, the density of the growth sites imaged by AFM is in excellent agreement with a calculation using the crystallization free energy. With lysozyme, the entropy effect due to the restructuring of the water molecules is negative. This leads to higher solubility.

Temperature-independent solubility and interactions between apoferritin monomers and dimers in solution

Abstract We used chromatographic, static and dynamic light scattering techniques, and atomic force microscopy (AFM) to study the structure of the protein species and the protein–protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of NaAc buffer and CdSO 4 . The sizes and shapes of the monomers and dimers, separated by size-exclusion chromatography, were determined by dynamic light scattering and AFM. While the monomer is an apparent sphere with a diameter corresponding to previous X-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was used to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The addition of even small amounts of Cd 2+ causes attraction between the monomer molecules. Furthermore, we found that the second virial coefficient and the protein solubility do not noticeably depend on temperature in the range from 0°C to 40°C. This suggests that the enthalpy for crystallization of apoferritin is close to zero, and the gain of entropy is essentially constant in this temperature range. We also found that in solutions of the apoferritin dimer, the molecules attract even in the presence of acetate buffer only, indicating a change in the surface of the apoferritin molecule. In view of the repulsion between the monomers at the same conditions, this suggests that the dimers and higher oligomers form only after partial unfolding of some of the apoferritin subunits. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization in the protein solution.

Molecular mechanisms of microheterogeneity‐induced defect formation in ferritin crystallization

We apply in situ atomic force microscopy to the crystallization of ferritins from solutions containing ≈5% (w/w) of their inherent molecular dimers. Molecular resolution imaging shows that the dimers consist of two bound monomers. The constituent monomers are likely partially denatured, resulting in increased hydrophobicity of the dimer surface. Correspondingly, the dimers strongly adsorb on the crystal surface. The adsorbed dimers hinder step growth and on incorporation by the crystal initiate stacks of up to 10 triple and single vacancies in the subsequent crystal layers. The molecules around the vacancies are shifted by ≈0.1 molecular dimensions from their crystallographic positions. The shifts strain the lattice and, as a consequence, at crystal sizes > 200 μm, the accumulated strain is resolved by a plastic deformation whereupon the crystal breaks into mosaic blocks 20–50 μm in size. The critical size for the onset of mosaicity is similar for ferritin and apoferritin and close to the value for a third protein, lysozyme; it also agrees with theoretical predictions. Trapped microcrystals in ferritin and apoferritin induce strain with a characteristic length scale equal to that of a single point defect, and, as a consequence, trapping does not contribute to the mosaicity. The sequence of undesired phenomena that include heterogeneity generation, adsorption, incorporation, and the resulting lattice strain and mosaicity in this and other proteins systems, could be avoided by improved methods to separate similar proteins species (microheterogeneity) or by increasing the biochemical stability of the macromolecules against oligomerization. Proteins 2001;43:343–352.

Tunneling spectroscopy and spectroscopic imaging of granular metallicity of polyaniline

The band-gap electronic structures of two forms of the conducting polymer polyaniline, emeraldine base (EB) and emeraldine salt (ES), have been studied. Tunneling spectroscopy measurements show that ES is metallic in nature with a finite electron density of state at the Fermi energy (EF), while EB behaves as an insulator with a gap at EF in agreement with theoretical calculations. These results allowed us to obtain direct evidence of the granular metallicity of ES by spectroscopic imaging. The spectroscopic images show different spatial distributions of nanometer-sized metallic particles in ES and EB samples, providing important information needed in studying electrical conductivities of conducting polymers.

An atomic force microscopy study of weathering of polyester/melamine paint surfaces

The use of an atomic force microscope (AFM) for rapid assessment of the durability of exterior polyester/melamine paints has been investigated. The study established an AFM imaging technique that produces data representative of weathering rates of paint films under a range of weathering regimes of varying severity. The effect of scan size on the average roughness parameter was investigated, leading to the adoption of two specified scan sizes. It was further found that both TappingMode™ and contact mode imaging gave equivalent detail of surface topography for these samples. However, TappingMode™ gave a clearer representation of the pigment/binder composite structure. Importantly, a technique was developed which reduces sample to sample variability by allowing a specified area to be repeatedly imaged as a function of weathering time. The images collected were found to show important detail regarding the mechanism of weathering, and the role of binder stability and pigment loss.

A prototype protein field-effect transistor

Electrical conduction in a macroscopic assembly of apoferritin, a non-redox protein, has been characterized using a three-terminal prototype device. Our result shows an ohmic conduction near zero bias. The ohmic conduction can be controlled using an electric field applied to the protein assembly via the gate terminal of the device. The transconductance of the protein device shows a highly nonlinear dependence on the gate voltage. The transconductance curve indicates that the device has the attributes of an n-channel metal-oxide-semiconductor field-effect transistor with electrons as charge carriers. The input/output dynamic response of the device has been demonstrated.

Enzyme-immobilized SiO2–Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s−1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

Are protein crystallization mechanisms relevant to understanding and control of polymerization of deoxyhemoglobin S

Abstract We investigated the homogeneous nucleation and subsequent evolution of polymers of sickle cell hemoglobin (HbS) using differential interference contrast (DIC) microscopy. The same technique was employed to determine the liquid–liquid separation boundaries for a variety of conditions in solution of sickle cell and normal human hemoglobin. The HbS polymers were also imaged using atomic force microscopy. We found that the location of Liquid–Liquid phase boundary under conditions that mimic those in the erythrocytes is consistent with previous determinations of the spinodal for this phase transition. Increasing the ionic strength shifts this phase boundary to significantly lower temperatures and Hb concentrations. We also found that the nucleation of individual HbS fibers indicates that the process is random and follows statistics similar to those established for nucleation of crystals or liquid droplets from vapors.