Ligand-Binding Cooperativity Effects in Polymer-Protein Conjugation.

We present an electron paramagnetic resonance (EPR) spectroscopic characterization of structural and dynamic effects that stem from post-translational modifications of bovine serum albumin (BSA), an established model system for polymer-protein conjugation. Beyond the typical drug delivery and biocompatibility aspect of such systems, we illustrate the causes that alter internal dynamics and therefore functionality in terms of ligand-binding to the BSA protein core. Uptake of the paramagnetic fatty acid derivative 16-doxyl stearic acid by several BSA-based squaric acid macroinitiators and polymer-protein conjugates was studied by EPR spectroscopy, aided by dynamic light scattering (DLS) and zeta potential measurements. The conjugates were grafted from oligo(ethylene glycol) methyl ether methacrylate (OEGMA), forming an overall core-shell-like structure. It is found that ligand-binding and associated parameters such as binding affinity, cooperativity, and the number of binding sites of BSA change drastically with the extent of surface modification. In the course of processing BSA, the ligands also change their preference for individual binding sites, as observed from a comparative view of their spatial alignments in double electron electron resonance (DEER) experiments. The protein-attached polymers constitute a diffusion barrier that significantly hamper ligand uptake. Moreover, zeta potentials (ζ) decrease linearly with the degree of surface modification in protein macroinitiators and an effective dielectric constant can be estimated for the polymer layer in the conjugates. All this suggests that ligand uptake characteristics in BSA can be fine-tuned by the extent and nature of such post-translational modifications (PTMs). We show that EPR spectroscopy is suitable for quantifying these subtle PTM-based functional effects from self-assembly of substrate and ligand.

Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin-Labeling and EPR Spectroscopy.

Drug binding to human serum albumin (HSA) has been characterized by a spin-labeling and continuous-wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin-binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR-active nitroxide radicals (spin-labeled pharmaceuticals (SLPs)) and in a screening approach CW-EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW-EPR spectra allow extraction of association constants (KA ) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug-protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged.

Using bound fatty acids to disclose the functional structure of serum albumin.

BACKGROUND: Serum albumin is a major transport protein in mammals and is known to have at least seven binding sites for long-chain fatty acids (FAs). SCOPE OF REVIEW: We have devised a new electron paramagnetic resonance (EPR) spectroscopic approach to gain information on the functional structure of serum albumin in solution in a "coarse-grained" manner from the ligands' point of view. Our approach is based on using spin labeled (paramagnetic) stearic acids self-assembled with albumin and subsequent nanoscale distance measurements between the FAs using double electron-electron resonance spectroscopy (DEER). Simple continuous wave (CW) EPR spectroscopy, which allows for quantification of bound ligands, complements our studies. MAJOR CONCLUSIONS: Based on DEER nanoscale distance measurements, the functional solution structure of human serum albumin (HSA) has remarkably been found to have a much more symmetric distribution of entry points to the FA binding sites than expected from the crystal structure, indicating increased surface flexibility and plasticity for HSA in solution. In contrast, for bovine serum albumin (BSA), the entry point topology is in good agreement with that expected from the crystal structure of HSA. Changes in the solution structures between albumins can hence be revealed and extended to more albumins to detect functional differences at the nanoscale. Going beyond fundamental structural studies, our research platform is also excellently suited for general studies of protein-solvent interactions, temperature effects and ligand binding. GENERAL SIGNIFICANCE: We discuss how our research platform helps illuminate protein dynamics and function and can be used to characterize albumin-based hybrid materials. This article is part of a Special Issue entitled Serum Albumin.

Dendronized albumin core-shell transporters with high drug loading capacity.

We describe the synthesis of a core-shell biohybrid consisting of a human serum albumin (HSA) core that serves as a reservoir for lipophilic molecules and a cationized shell region consisting of ethynyl-G2.0-PAMAM or ethynyl-G3.0-PAMAM dendrons. The binding capacity of lipophilic guests was quantified applying electron paramagnetic resonance (EPR) spectroscopy, and five to six out of seven pockets were still available compared with HSA. The attachment of ethynyl-G2.0-PAMAM dendrons to HSA yielded a nontoxic core-shell macromolecule that was clearly uptaken by A549 human epithelial cells due to the presence of the dendritic PAMAM shell. Significantly higher loading of doxorubicin was observed for dendronized G2-DHSA compared with the native protein due to the availability of binding pockets of the HSA core, and interaction with the dendritic shell. Dendronized G2-DHSA-doxorubicin displayed significant cytotoxicity resulting from high drug loading and high stability under different conditions, thus demonstrating its great potential as a transporter for drug molecules.

Glycation Alters the Fatty Acid Binding Capacity of Human Serum Albumin.

Glycation significantly alters the physicochemical and biofunctional properties of proteins in foods and in vivo. In the present study, human serum albumin (HSA) as the major transporter of fatty acids was modified with glyoxal under physiological conditions. Reversibly albumin-bound glyoxal was removed, and advanced glycation end products were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total modification of protein-bound lysine and arginine residues reached up to 4.2 and 9.6%, respectively. The impact of these modifications on the transport capacity of long-chain fatty acids was characterized by spin-labeled fatty acid probes via electron paramagnetic resonance spectroscopy. With increasing degree of glycation, the equivalence of the seven binding sites of native HSA with a dissociation constant of 0.74 ± 0.09 µM was set off with only the three high-affinity sites 2, 4, and 5 remaining (0.46 ± 0.07 µM). The other four sites were shifted to low affinities with significantly higher dissociation constants (1.32 ± 0.35 µM). Tryptic peptide mapping enabled us to relate these findings to molecular changes at specific binding sites. Modification hotspots identified were lysine 351, 286, 159 and arginine 144, 485, 117. Further investigation of plasma protein samples of uremic patients vs healthy controls gave first insights into the in vivo situation.

Low pH structure of heliorhodopsin reveals chloride binding site and intramolecular signaling pathway.

Within the microbial rhodopsin family, heliorhodopsins (HeRs) form a phylogenetically distinct group of light-harvesting retinal proteins with largely unknown functions. We have determined the 1.97 Å resolution X-ray crystal structure of Thermoplasmatales archaeon SG8-52-1 heliorhodopsin (TaHeR) in the presence of NaCl under acidic conditions (pH 4.5), which complements the known 2.4 Å TaHeR structure acquired at pH 8.0. The low pH structure revealed that the hydrophilic Schiff base cavity (SBC) accommodates a chloride anion to stabilize the protonated retinal Schiff base when its primary counterion (Glu-108) is neutralized. Comparison of the two structures at different pH revealed conformational changes connecting the SBC and the extracellular loop linking helices A-B. We corroborated this intramolecular signaling transduction pathway with computational studies, which revealed allosteric network changes propagating from the perturbed SBC to the intracellular and extracellular space, suggesting TaHeR may function as a sensory rhodopsin. This intramolecular signaling mechanism may be conserved among HeRs, as similar changes were observed for HeR 48C12 between its pH 8.8 and pH 4.3 structures. We additionally performed DEER experiments, which suggests that TaHeR forms possible dimer-of-dimer associations which may be integral to its putative functionality as a light sensor in binding a transducer protein.

Electron paramagnetic resonance spectroscopy on G-protein-coupled receptors: Adopting strategies from related model systems.

Membrane proteins, including ion channels, transporters and G-protein-coupled receptors (GPCRs), play a significant role in various physiological processes. Many of these proteins are difficult to express in large quantities, imposing crucial experimental restrictions. Nevertheless, there is now a wide variety of studies available utilizing electron paramagnetic resonance (EPR) spectroscopic techniques that expand experimental accessibility by using relatively small quantities of protein. Here, we give an overview starting from basic strategies in EPR on membrane proteins with a focus on GPCRs, while emphasizing several applications from recent years. We highlight how the arsenal of EPR-based techniques may provide significant further contributions to understanding the complex molecular machinery and energetic phenomena responsible for seamless workflow in essential biological processes.

Probing the Nanoscopic Thermodynamic Fingerprint of Paramagnetic Ligands Interacting with Amphiphilic Macromolecules.

Self-assembly of macromolecules with ligands is an intricate dynamic process that depends on a wide variety of parameters and forms the basis of many essential biological processes. We elucidate the underlying energetic processes of self-assembly in a model system consisting of amphiphilic core-shell polymers interacting with paramagnetic, amphiphilic ligand molecules from temperature-dependent continuous wave electron paramagnetic resonance (CW EPR) spectroscopy subsequent to spectral simulation. The involved processes as observed from the ligands' point of view are either based on temperature-dependent association constants (KA,j,k) or dynamic rotational regime interconversion (IC) constants (KIC,j,k). The interconversion process describes a transition from Brownian (b1) towards free (b2) diffusion of ligand. Both processes exhibit non-linear van't Hoff (lnK vs. T-1) plots in the temperature range of liquid water and we retrieve decisive dynamic information of the system from the energetic fingerprints of ligands on the nanoscale, especially from the temperature-dependent interconversion heat capacity (∆C°P,IC).

Modeling excluded volume effects for the faithful description of the background signal in double electron-electron resonance.

We discuss excluded volume effects on the background signal of double electron-electron resonance (DEER) experiments. Assuming spherically symmetric pervaded volumes, an analytical expression of the background signal is derived based on the shell-factorization approach. The effects of crowding and off-center label positions are discussed. Crowding is taken into account using the Percus-Yevick approximation for the radial distribution function of the particle centers. In addition, a versatile approach relating the pair-correlation function of the particle centers with those of off-center labels is introduced. Limiting expressions applying to short and long dipolar evolution times are derived. Furthermore, we show under which conditions the background with significant excluded volume effects resembles that originating from a fractal dimensionality ranging from 3 to 6. DEER time domain data of spin-probed samples of human serum albumin (HSA) are shown to be strongly affected by excluded-volume effects. The excluded volume is determined from the simultaneous analysis of spectra recorded at various protein concentrations but a constant probe-to-protein ratio. The spin-probes 5-DOXYL-stearic acid (5-DSA) and 16-DOXYL-stearic acid (16-DSA) are used, which, when taken up by HSA, give rise to broad and well-defined distance distributions, respectively. We compare different, model-free approaches of analyzing these data. The most promising results are obtained by the concurrent Tikhonov regularization of all spectra when a common background model is simultaneously adjusted such that the a posteriori probability is maximized. For the samples of 16-DSA in HSA, this is the only approach that allows suppressing a background artifact. We suggest that the delineated simultaneous analysis procedure can be generally applied to reduce ambiguities related to the ill-posed extraction of distance distributions from DEER spectra. This approach is particularly valuable for dipolar signals resulting from broad distance distributions, which as a consequence, are devoid of explicit dipolar oscillations.

Evidence for water-tuned structural differences in proteins: an approach emphasizing variations in local hydrophilicity.

We present experimental evidence for the significant effect that water can have on the functional structure of proteins in solution. Human (HSA) and Bovine Serum Albumin (BSA) have an amino acid sequence identity of 75.52% and are chosen as model proteins. We employ EPR-based nanoscale distance measurements using double electron-electron resonance (DEER) spectroscopy and both albumins loaded with long chain fatty acids (FAs) in solution to globally (yet indirectly) characterize the tertiary protein structures from the bound ligands' points of view. The complete primary structures and crystal structures of HSA and as of recently also BSA are available. We complement the picture as we have recently determined the DEER-derived solution structure of HSA and here present the corresponding BSA solution structure. The characteristic asymmetric FA distribution in the crystal structure of HSA can surprisingly be observed by DEER in BSA in solution. This indicates that the BSA conformational ensemble in solution seems to be narrow and close to the crystal structure of HSA. In contrast, for HSA in solution a much more symmetric FA distribution was found. Thus, conformational adaptability and flexibility dominate in the HSA solution structure while BSA seems to lack these properties. We further show that differences in amino acid hydropathies of specific structural regions in both proteins can be used to correlate the observed difference in the global (tertiary) solution structures with the differences on the primary structure level.