Eva Thulin

Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles

Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle “surface,” leading to a protein “corona” that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein–nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.

Nucleation of protein fibrillation by nanoparticles

Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimers, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleation-dependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human β2-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and β2-microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surface-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.

Inhibition of Amyloid beta Protein Fibrillation by Polymeric Nanoparticles

Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimers disease-associated amyloid beta protein (Abeta). We found that these nanoparticles affect mainly the nucleation step of Abeta fibrillation. The elongation step is largely unaffected by the particles, and once the Abeta is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of Abeta is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that Abeta binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric Abeta and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of Abeta initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear.

Calcium-induced structural changes and domain autonomy in calmodulin.

We have determined the solution structures of the apo and (Ca2+)2 forms of the carboxy-terminal domain of calmodulin using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The results show that both forms adopt well-defined structures with essentially equal secondary structure. A comparison of the structures of the two forms shows that Ca2+ binding causes major rearrangements of the secondary structure elements with changes in inter-residue distances of up to 15 Å and exposure of the hydrophobic interior of the four-helix bundle. Comparisons with previously determined high-resolution X-ray structures and models of calmodulin indicate that this domain is structurally autonomous.

A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide.

We report the development of a high‐level bacterial expression system for the Alzheimer’s disease‐associated amyloid β‐peptide (Aβ), together with a scaleable and inexpensive purification procedure. Aβ(1–40) and Aβ(1–42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met‐Aβ(1–40) and Met‐Aβ(1–42), referred to as Aβ(Μ1–40) and Aβ(Μ1–42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion‐exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Aβ peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Aβ1–40 and Aβ1–42. Formation of fibrils by all peptides was concentration‐dependent, and exhibited kinetics typical of a nucleation‐dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.

Proline cis-trans isomers in calbindin D9k observed by X-ray crystallography☆

In a structure of recombinant bovine calbindin D9k, determined crystallographically to 1.6 A resolution, a proline in mixed, approximately equally populated, cis and trans conformation is observed. Isomers of this kind have not been reported in structure determinations of calbindin D9k to 2.3 A resolution or in any other crystallographically determined protein structure. The cis-trans isomerization occurs at the peptide bond between Gly42 and Pro43, which is in agreement with results from two-dimensional 1H nuclear magnetic resonance spectroscopy experiments on solutions of calbindin D9k. Alternative backbone stretches have been modeled and refined by stereochemical restrained least-squares refinement for the segment Lys41 to Pro43. The final R-value was 0.188. The structural perturbations accompanying the cis-trans isomerization are found to be very localized. The largest positional differences are observed at residue Gly42, in which the alternative positions of the oxygen atom are 3.6 A apart.

A 113Cd NMR study of calmodulin and its interaction with calcium, magnesium and trifluoperazine

Calcium has been long recognized as an important regulator of a variety of cellular events [l-5]. The detailed mechanism whereby calcium acts is still largely unknown. However, it has been demonstrated that calcium regulation of a number of enzyme systems is mediated by a low molecular weight, thermostable protein termed calmodulin. This protein was first described [4-7] as an activator of brain cyclic nudeotide phosphodiesterase. Cahnodulin has subsequently been found in tissues from various organs in both vertebrate, invertebrate and plant species [3,8,9,9a]. There is good evidence that calmoduliu represents an ubiquitous calcium regulatory protein whose primary sequence is highly conserved throughout all eucaryotic cells 133. Calmodulin (M, 16 700) consists of a single polypeptide chain whose amino acid composition is characterized by having a large number of acidic residues (glutamic and aspartic), a lack of cysteine and tryptophan, and the presence of one mole of the unusual amino acid trimethyllysine [lo]. Calmoduhn’s sequence is homologous with that of parvalbumin and skeletal troponin C (TnC) and like the latter its ammo acid sequence can be divided into 4 internally homologous domains, each of which has a potent~l calcium binding site [ 10 J. The calcium binding properties of calmodulin have been subject to several studies and while there is some inconsistency as regards the relative number of high and low affinity sites most studies indicate that calmodulin will bind 4 mol calciumlmol protein [ 1115 ] . Expe~ment~ evidence from a number of studies indicate that calcium binding to calmodulin is accompanied by pronounced changes in its solution conformation [ 1 l-14,16-22].

Calbindin D28k Exhibits Properties Characteristic of a Ca2+ Sensor

Calbindin D28k is a member of the calmodulin superfamily of Ca2+-binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca2+ buffer, but the studies presented in this work indicate that it may also act as a Ca2+ sensor. The results show that Mg2+ binds to the same sites as Ca2+with an association constant of ∼1.4·103 m −1 in 0.15 m KCl. The four high affinity sites in calbindin D28k bind Ca2+ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg2+, the Ca2+ affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca2+ concentration in a resting cell calbindin D28k is saturated to 40–75% with Mg2+ but to less than 9% with Ca2+. In contrast, the protein is expected to be nearly fully saturated with Ca2+ at the Ca2+ level of an activated cell. A substantial conformational change is observed upon Ca2+ binding, but only minor structural changes take place upon Mg2+ binding. This suggests that calbindin D28k undergoes Ca2+-induced structural changes upon Ca2+activation of a cell. Thus, calbindin D28k displays several properties that would be expected for a protein involved in Ca2+-induced signal transmission and hence may function not only as a Ca2+ buffer but also as a Ca2+sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca2+, becomes exposed upon Ca2+binding.

Calcium-dependent hydrophobic interaction chromatography of calmodulin, troponin C and their proteolytic fragments

The homologous calcium‐binding proteins calmodulin and skeletal and cardiac troponin C bind in the presence of 1 mM Ca2+ to phenyl‐Sepharose and can be eluted in buffers containing chelators. Results obtained with a series of proteolytic fragments, which were prepared by limited tryptic or thrombic degradation, showed that this Ca2+‐dependent hydrophobic interaction chromatography provides a convenient method for the large scale purification of some of these peptides. Moreover, it was found that in troponin C only one Ca2+‐induced hydrophobic site is located in the amino‐terminal half of the protein, but that calmodulin contained such sites in both the amino‐ and carboxy‐terminal halves of the molecule. The probable location of the latter site is discussed.

The EF-hand domain: a globally cooperative structural unit.

EF‐hand Ca2+‐binding proteins participate in both modulation of Ca2+ signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca2+. The EF‐hand domains of calbindin D9k and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium‐induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF‐hand protein calbindin D9k to alter the packing of helices I and II in the apoprotein. The three‐dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long‐range effects provides clear evidence that four‐helix EF‐hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long‐range effects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a proteins fold and function.