Small angle X-ray scattering and molecular dynamic simulations provide molecular insight for stability of recombinant human transferrin.

Transferrin is an attractive candidate for drug delivery due to its ability to cross the blood brain barrier. However, in order to be able to use it for therapeutic purposes, it is important to investigate how its stability depends on different formulation conditions. Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes. Lowering pH induces opening of the transferrin N-lobe, which results in a negative effect on the stability. Presence of NaCl or arginine at low pH enhances the opening and has a negative impact on the overall protein stability. STATEMENT OF SIGNIFICANCE: Protein-based therapeutics have become an essential part of medical treatment. They are highly specific, have high affinity and fewer off-target effects. However, stabilization of proteins is critical, time-consuming, and expensive, and it is not yet possible to predict the behavior of proteins under different conditions. The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and therefore with high potential in drug delivery. Combination of high throughput unfolding techniques and structural studies, using small angle X-ray scattering and molecular dynamic simulations, allows us to understand the behavior of transferrin on a molecular level.

Concentrated protein solutions investigated using acoustic levitation and small-angle X-ray scattering.

An acoustically levitated droplet has been used to collect synchrotron SAXS data on human serum albumin protein solutions up to a protein concentration of 400 mg ml-1. A careful selection of experiments allows for fast data collection of a large amount of data, spanning a protein concentration/solvent concentration space with limited sample consumption (down to 3 µL per experiment) and few measurements. The data analysis shows data of high quality that are reproducible and comparable with data from standard flow-through capillary-based experiments. Furthermore, using this methodology, it is possible to achieve concentrations that would not be accessible by conventional cells. The protein concentration and ionic strength parameter space diagram may be covered easily and the amount of protein sample is significantly reduced (by a factor of 100 in this work). Used in routine measurements, the benefits in terms of protein cost and time spent are very significant.

Investigations of Albumin-Insulin Detemir Complexes Using Molecular Dynamics Simulations and Free Energy Calculations.

Insulin detemir is a lipidated insulin analogue that obtains a half-life extension by oligomerization and reversible binding to human serum albumin. In the present study, the complex between a detemir hexamer and albumin is investigated by an integrative approach combining molecular dynamics (MD) simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations, and dynamic light scattering (DLS) experiments. Recent reported small-angle X-ray scattering data could not unambiguously resolve the exact binding site of detemir on albumin. We therefore applied MD simulations to deduce the binding site and key protein-protein interactions. MD simulations were started from initial complex structures based on the SAXS models, and free energies of binding were estimated from the simulations by using the MM-PBSA approach for the different binding positions. The results suggest that the overlapping FA3-FA4 binding site (named FA4) is the most favorable site with a calculated free energy of binding of -28 ± 6 kcal/mol and a good fit to the reported SAXS data throughout the simulations. Multiple salt bridges, hydrogen bonds, and favorable van der Waals interactions are observed in the binding interface that promote complexation. The binding to FA4 is further supported by DLS competition experiments with the prototypical FA4 ligand, ibuprofen, showing displacement of detemir by ibuprofen. This study provides information on albumin-detemir binding on a molecular level, which could be utilized in a rational design of future lipidated albumin-binding peptides.

Solution structures of long-acting insulin analogues and their complexes with albumin.

The lipidation of peptide drugs is one strategy to obtain extended half-lives, enabling once-daily or even less frequent injections for patients. The half-life extension results from a combination of self-association and association with human serum albumin (albumin). The self-association and association with albumin of two insulin analogues, insulin detemir and insulin degludec, were investigated by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) in phenolic buffers. Detemir shows concentration-dependent self-association, with an equilibrium between hexamer, dihexamer, trihexamer and larger species, while degludec appears as a dihexamer independent of concentration. The solution structure of the detemir trihexamer has a bent shape. The stoichiometry of the association with albumin was studied using DLS. For albumin-detemir the molar stoichiometry was determined to be 1:6 (albumin:detemir ratio) and for albumin-degludec it was between 1:6 and 1:12 (albumin:degludec ratio). Batch SAXS measurements of a 1:6 albumin:detemir concentration series revealed a concentration dependence of complex formation. The data allowed the modelling of a complex between albumin and a detemir hexamer and a complex consisting of two albumins binding to opposite ends of a detemir dihexamer. Measurements of size-exclusion chromatography coupled to SAXS revealed a complex between a degludec dihexamer and albumin. Based on the results, equilibria for the albumin-detemir and albumin-degludec mixtures are proposed.

Self-Interaction of Human Serum Albumin: A Formulation Perspective.

In the present study, small-angle X-ray scattering (SAXS) and static light scattering (SLS) have been used to study the solution properties and self-interaction of recombinant human serum albumin (rHSA) molecules in three pharmaceutically relevant buffer systems. Measurements are carried out up to high protein concentrations and as a function of ionic strength by adding sodium chloride to probe the role of electrostatic interactions. The effective structure factors (S eff) as a function of the scattering vector magnitude q have been extracted from the scattering profiles and fit to the solution of the Ornstein-Zernike equation using a screened Yukawa potential to describe the double-layer force. Although only a limited q range is used, accurate fits required including an electrostatic repulsion element in the model at low ionic strength, while only a hard sphere model with a tunable diameter is necessary for fitting to high-ionic-strength data. The fit values of net charge agree with available data from potentiometric titrations. Osmotic compressibility data obtained by extrapolating the SAXS profiles or directly from SLS measurements has been fit to a 10-term virial expansion for hard spheres and an equation of state for hard biaxial ellipsoids. We show that modeling rHSA as an ellipsoid, rather than a sphere, provides a much more accurate fit for the thermodynamic data over the entire concentration range. Osmotic virial coefficient data, derived at low protein concentration, can be used to parameterize the model for predicting the behavior up to concentrations as high as 450 g/L. The findings are especially important for the biopharmaceutical sector, which require approaches for predicting concentrated protein solution behavior using minimal sample consumption.

Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life.

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though GLP-1 is interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of <3 min limits its clinical use. A strategy for extending the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days because of its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial cells of blood vessels, which rescues circulating HSA from lysosomal degradation. We have conjugated GLP-1 to C34 of native sequence recombinant HSA (rHSA) and two rHSA variants, one with increased and one with decreased binding affinity for human FcRn. We have investigated the impact of conjugation on FcRn binding affinities, GLP-1 potency, and pharmacokinetics, combined with the solution structure of the rHSA variants and GLP-1-albumin conjugates. The solution structures, determined by small-angle X-ray scattering, show the GLP-1 pointing away from the surface of rHSA. Combining the solution structures with the available structural information about the FcRn and GLP-1 receptor obtained from X-ray crystallography, we can explain the observed in vitro and in vivo behavior. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease in the potency of GLP-1 can be explained by a steric hindrance of binding of GLP-1 to its receptor.

Small-Angle X-ray Scattering Data in Combination with RosettaDock Improves the Docking Energy Landscape.

We have performed a benchmark to evaluate the relative success of using small-angle X-ray scattering (SAXS) data as constraints (hereafter termed SAXSconstrain) in the RosettaDock protocol (hereafter termed RosettaDockSAXS). For this purpose, we have chosen 38 protein complex structures, calculated the theoretical SAXS data for the protein complexes using the program CRYSOL, and then used the SAXS data as constraints. We further considered a few examples where crystal structures and experimental SAXS data are available. SAXSconstrain were added to the protocol in the initial, low-resolution docking step, allowing fast rejection of complexes that violate the shape restraints imposed by the SAXS data. Our results indicate that the implementation of SAXSconstrain in general reduces the sampling space of possible protein-protein complexes significantly and can indeed increase the probability of finding near-native protein complexes. The methodology used is based on rigid-body docking and works for cases where no or minor conformational changes occur upon binding of the docking partner. In a wider perspective, the strength of RosettaDockSAXS lies in the combination of low-resolution structural information on protein complexes in solution from SAXS experiments with protein-protein interaction energies obtained from RosettaDock, which will allow the prediction of unknown three-dimensional atomic structures of protein-protein complexes.

Insulin fibrillation: The influence and coordination of Zn2.

Protein amyloid fibrillation is obtaining much focus because it is connected with amyloid-related human diseases such as Alzheimer's disease, diabetes mellitus type 2, or Parkinson's disease. The influence of metal ions on the fibrillation process and whether it is implemented in the amyloid fibrils has been debated for some years. We have therefore investigated the influence and binding geometry of zinc in fibrillated insulin using extended X-ray absorption fine-structure and X-ray absorption near-edge structure spectroscopy. The results were validated with fibre diffraction, Transmission Electron Microscopy and Thioflavin T fluorescence measurements. It is well-known that Zn2+ ions coordinate and stabilize the hexameric forms of insulin. However, this study is the first to show that zinc indeed binds to the insulin fibrils. Furthermore, zinc influences the kinetics and the morphology of the fibrils. It also shows that zinc coordinates to histidine residues in an environment, which is similar to the coordination seen in the insulin R6 hexamers, where three histidine residues and a chloride ion is coordinating the zinc.

Oligomerization of a Glucagon-like Peptide 1 Analog: Bridging Experiment and Simulations.

The glucagon-like peptide 1 (GLP-1) analog, liraglutide, is a GLP-1 agonist and is used in the treatment of type-2 diabetes mellitus and obesity. From a pharmaceutical perspective, it is important to know the oligomerization state of liraglutide with respect to stability. Compared to GLP-1, liraglutide has an added fatty acid (FA) moiety that causes oligomerization of liraglutide as suggested by small-angle x-ray scattering (SAXS) and multiangle static light scattering (MALS) results. SAXS data suggested a global shape of a hollow elliptical cylinder of size hexa-, hepta-, or octamer, whereas MALS data indicate a hexamer. To elaborate further on the stability of these oligomers and the role of the FA chains, a series of molecular-dynamics simulations were carried out on 11 different hexa-, hepta-, and octameric systems. Our results indicate that interactions of the fatty acid chains contribute noticeably to the stabilization. The simulation results indicate that the heptamer with paired FA chains is the most stable oligomer when compared to the 10 other investigated structures. Theoretical SAXS curves extracted from the simulations qualitatively agree with the experimentally determined SAXS curves supporting the view that liraglutide forms heptamers in solution. In agreement with the SAXS data, the heptamer forms a water-filled oligomer of elliptical cylindrical shape.

The intrinsically disordered RNR inhibitor Sml1 is a dynamic dimer.

Sml1 is a small ribonucleotide reductase (RNR) regulatory protein in Saccharomyces cerevisiae that binds to and inhibits RNR activation. NMR studies of 15N-labeled Sml1 (104 residues), as well as of a truncated variant (residues 50-104), have allowed characterization of their molecular properties. Sml1 belongs to the class of intrinsically disordered proteins with a high degree of dynamics and very little stable structure. Earlier suggestions for a dimeric structure of Sml1 were confirmed, and from translation diffusion NMR measurements, a dimerization dissociation constant of 0.1 mM at 4 degreesC could be determined. The hydrodynamic radius for the monomeric form of Sml1 was determined to be 23.4 A, corresponding to a protein size between those of a globular protein and a coil. Formation of a dimer results in a hydrodynamic radius of 34.4 A. The observed chemical shifts showed in agreement with previous studies two segments with transient helical structure, residues 4-20 and 60-86, and relaxation studies clearly showed restricted motion in these segments. A spin-label attached to C14 showed long-range interactions with residues 60-70 and 85-95, suggesting that the N-terminal domain folds onto the C-terminal domain. Importantly, protease degradation studies combined with mass spectrometry indicated that the N-terminal domain is degraded before the C-terminal region and thus may serve as a protection against proteolysis of the functionally important C-terminal region. Dimer formation was not associated with significant induction of structure but was found to provide further protection against proteolysis. We propose that this molecular shielding and protection of vital functional structures from degradation by functionally unimportant sites may be a general attribute of other natively disordered proteins.