Microheterogeneity in frozen protein solutions.
Ontology highlight
ABSTRACT:
SUBMITTER: Twomey A
PROVIDER: S-EPMC4430435 | biostudies-literature | 2015 Jun
REPOSITORIES: biostudies-literature
Publications
Microheterogeneity in frozen protein solutions.
Twomey Alan A Kurata Kosaku K Nagare Yutaka Y Takamatsu Hiroshi H Aksan Alptekin A
International journal of pharmaceutics 20150415 1-2
In frozen and lyophilized systems, the biological to be stabilized (e.g. therapeutic protein, biomarker, drug-delivery vesicle) and the cryo-/lyo-protectant should be co-localized for successful stabilization. During freezing and drying, many factors cause physical separation of the biological from the cryo-/lyo-protectant, called microheterogeneity (MH), which may result in poor stabilization efficiency. We have developed a novel technique that utilized confocal Raman microspectroscopy in combi ...[more]
Similar Datasets
Project description:There is currently great interest in understanding the limits on NMR signal enhancements provided by dynamic nuclear polarization (DNP), and in particular if the theoretical maximum enhancements can be achieved. We show that over a 2-fold improvement in cross-effect DNP enhancements can be achieved in MAS experiments on frozen solutions by simply incorporating solid particles into the sample. At 9.4 T and ?105 K, enhancements up to ?H = 515 are obtained in this way, corresponding to 78% of the theoretical maximum. We also underline that degassing of the sample is important to achieve highest enhancements. We link the amplification effect to the dielectric properties of the solid material, which probably gives rise to scattering, diffraction, and amplification of the microwave field in the sample. This is substantiated by simulations of microwave propagation. A reduction in sample heating at a given microwave power also likely occurs due to reduced dielectric loss. Simulations indicate that the microwave field (and thus the DNP enhancement) is inhomogeneous in the sample, and we deduce that in these experiments between 5 and 10% of the solution actually yields the theoretical maximum signal enhancement of 658. The effect is demonstrated for a variety of particles added to both aqueous and organic biradical solutions.
| S-EPMC4671102 | biostudies-literature
Project description:The effects of nuclear signal quenching induced by the presence of a paramagnetic polarizing agent are documented for conditions used in magic angle spinning (MAS)-dynamic nuclear polarization (DNP) experiments on homogeneous solutions. In particular, we present a detailed analysis of three time constants: (1) the longitudinal build-up time constant TB for (1)H; (2) the rotating frame relaxation time constant T1? for (1)H and (13)C and (3) T2 of (13)C, the transverse relaxation time constant in the laboratory frame. These relaxation times were measured during microwave irradiation at a magnetic field of 5 T (140 GHz) as a function of the concentration of four polarizing agents: TOTAPOL, 4-amino-TEMPO, trityl (OX063), and Gd-DOTA and are compared to those obtained for a sample lacking paramagnetic doping. We also report the EPR relaxation time constants T1S and T2S, the DNP enhancements, ?, and the parameter E, defined below, which measures the sensitivity enhancement for the four polarizing agents as a function of the electron concentration. We observe substantial intensity losses (paramagnetic quenching) with all of the polarizing agents due to broadening mechanisms and cross relaxation during MAS. In particular, the monoradical trityl and biradical TOTAPOL induce ?40% and 50% loss of signal intensity. In contrast there is little suppression of signal intensity in static samples containing these paramagnetic species. Despite the losses due to quenching, we find that all of the polarizing agents provide substantial gains in signal intensity with DNP, and in particular that the net enhancement is optimal for biradicals that operate with the cross effect. We discuss the possibility that much of this polarization loss can be regained with the development of instrumentation and methods to perform electron decoupling.
| S-EPMC3951579 | biostudies-literature
Project description:Electron spin-lattice relaxation of two trityl radicals, d24-OX063 and Finland trityl, were studied under conditions relevant to their use in dissolution dynamic nuclear polarization (DNP). The dependence of relaxation kinetics on temperature up to 100 K and on concentration up to 60 mM was obtained at X- and W-bands (0.35 and 3.5 Tesla, respectively). The relaxation is quite similar at both bands and for both trityl radicals. At concentrations typical for DNP, relaxation is mediated by excitation transfer and spin-diffusion to fast-relaxing centers identified as triads of trityl radicals that spontaneously form in the frozen samples. These centers relax by an Orbach-Aminov mechanism and determine the relaxation, saturation and electron spin dynamics during DNP.
| S-EPMC5482570 | biostudies-literature
Project description:Distance measurements using double electron-electron resonance (DEER) and Gd(3+) chelates for spin labels (GdSL) have been shown to be an attractive alternative to nitroxide spin labels at W-band (95GHz). The maximal distance that can be accessed by DEER measurements and the sensitivity of such measurements strongly depends on the phase relaxation of Gd(3+) chelates in frozen, glassy solutions. In this work, we explore the phase relaxation of Gd(3+)-DOTA as a representative of GdSL in temperature and concentration ranges typically used for W-band DEER measurements. We observed that in addition to the usual mechanisms of phase relaxation known for nitroxide based spin labels, GdSL are subjected to an additional phase relaxation mechanism that features an increase in the relaxation rate from the center to the periphery of the EPR spectrum. Since the EPR spectrum of GdSL is the sum of subspectra of the individual EPR transitions, we attribute this field dependence to transition dependent phase relaxation. Using simulations of the EPR spectra and its decomposition into the individual transition subspectra, we isolated the phase relaxation of each transition and found that its rate increases with |ms|. We suggest that this mechanism is due to transient zero field splitting (tZFS), where its magnitude and correlation time are scaled down and distributed as compared with similar situations in liquids. This tZFS induced phase relaxation mechanism becomes dominant (or at least significant) when all other well-known phase relaxation mechanisms, such as spectral diffusion caused by nuclear spin diffusion, instantaneous and electron spin spectral diffusion, are significantly suppressed by matrix deuteration and low concentration, and when the temperature is sufficiently low to disable spin lattice interaction as a source of phase relaxation.
| S-EPMC4495766 | biostudies-literature
Project description:BACKGROUND:In 2008, the US Food and Drug Administration (FDA) issued a Guidance for Industry statement formally recognizing (during drug development) the conjoined nature of type 2 diabetes (T2D) and cardiovascular disease (CVD), which has precipitated an urgent need for panels of markers (and means of analysis) that are able to differentiate subtypes of CVD in the context of T2D. Here, we explore the possibility of creating such panels using the working hypothesis that proteins, in addition to carrying time-cumulative marks of hyperglycemia (e.g., protein glycation in the form of Hb A(?c)), may carry analogous information with regard to systemic oxidative stress and aberrant enzymatic signaling related to underlying pathobiologies involved in T2D and/or CVD. METHODS:We used mass spectrometric immunoassay to quantify, in targeted fashion, relative differences in the glycation, oxidation, and truncation of 11 specific proteins. RESULTS:Protein oxidation and truncation (owing to modified enzymatic activity) are able to distinguish between subsets of diabetic patients with or without a history of myocardial infarction and/or congestive heart failure where markers of glycation alone cannot. CONCLUSION:Markers based on protein modifications aligned with the known pathobiologies of T2D represent a reservoir of potential cardiovascular markers that are needed to develop the next generation of antidiabetes medications.
| S-EPMC3761388 | biostudies-literature
Project description:NMR-Profiles are quantitative one-dimensional (1D) presentations of 2D [¹?N, ¹H]-correlation spectra used to monitor the quality of protein solutions prior to and during NMR structure determinations and functional studies. In our current use in structural genomics projects, an NMR-Profile is recorded at the outset of a structure determination, using a uniformly ¹?N-labeled microscale sample of the protein. We thus assess the extent to which polypeptide backbone resonance assignments can be achieved with given NMR techniques, for example, conventional triple resonance experiments or APSY-NMR. With the availability of sequence-specific polypeptide backbone resonance assignments in the course of the structure determination, an "Assigned NMR-Profile" is generated, which visualizes the variation of the ¹?N - ¹H correlation cross peak intensities along the sequence and thus maps the sequence locations of polypeptide segments for which the NMR line shapes are affected by conformational exchange or other processes. The Assigned NMR-Profile provides a guiding reference during later stages of the structure determination, and is of special interest for monitoring the protein during functional studies, where dynamic features may be modulated during physiological processes.
| S-EPMC3960936 | biostudies-literature
Project description:Protein aggregation is broadly important in diseases and in formulations of biological drugs. Here, we develop a theoretical model for reversible protein-protein aggregation in salt solutions. We treat proteins as hard spheres having square-well-energy binding sites, using Wertheim's thermodynamic perturbation theory. The necessary condition required for such modeling to be realistic is that proteins in solution during the experiment remain in their compact form. Within this limitation our model gives accurate liquid-liquid coexistence curves for lysozyme and ? IIIa-crystallin solutions in respective buffers. It provides good fits to the cloud-point curves of lysozyme in buffer-salt mixtures as a function of the type and concentration of salt. It than predicts full coexistence curves, osmotic compressibilities, and second virial coefficients under such conditions. This treatment may also be relevant to protein crystallization.
| S-EPMC4450416 | biostudies-literature
Project description:The microscopic structure of frozen aqueous sucrose solutions, over concentrations of 0-75% (w/v), is characterized by using multiple continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopic and relaxation techniques and the paramagnetic spin probe, TEMPOL. The temperature dependence of the TEMPOL EPR line-shape anisotropy reveals a mobility transition, specified at 205 K in pure water and 255 ± 5 K for >1% (w/v) added sucrose. The transition temperature is >>Tg, where Tg is the homogeneous water glass transition temperature, which shows that TEMPOL resides in the mesoscopic domain (mesodomain) at water-ice crystallite boundaries and that the mesodomain sucrose concentrations are comparable at >1% (w/v) added sucrose. Electron spin-echo envelope modulation (ESEEM) spectroscopy of TEMPOL-(2)H2-sucrose hyperfine interactions also indicates comparable sucrose concentrations in mesodomains at >1% (w/v) added sucrose. Electron spin-echo (ESE) detected longitudinal and phase memory relaxation times (T1 and TM, respectively) at 6 K indicate a general trend of increased mesodomain volume with added sucrose, in three stages: 1-15, 20-50, and >50% (w/v). The calibrated TEMPOL concentrations indicate that the mesodomain volume is less than the predicted maximally freeze-concentrated value [80 (w/w); 120% (w/v)], with transitions at 15-20% and 50% (w/v) starting sucrose. An ordered sucrose hydrate phase, which excludes TEMPOL, and a disordered, amorphous sucrose-water glass phase, in which TEMPOL resides, are proposed to compose a heterogeneous mesodomain. The results show that the ratio of ordered and disordered volume fractions in the mesodomain is exquisitely sensitive to the starting sucrose concentration.
| S-EPMC3623541 | biostudies-literature
Project description:Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein-protein, or protein-small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: ?1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin-hemoglobin (Hp-Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp-Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein-drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.
| S-EPMC6126716 | biostudies-literature
Project description:Continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy of highly-anisotropic low spin (HALS) ferric heme centers in frozen solutions is not a very informative approach because usually only one feature is reliably observed in the spectra, that at the maximal principal g-value of, typically, 3.3-3.79. The other two EPR turning points are severely broadened by g-strain and are not easily observed in the first-derivative CW EPR spectra. In this work, we have explored the potential of alternative EPR techniques, the electron spin echo (ESE) field sweep and electron spin transient nutation (TN), for obtaining information about the g-tensors of such systems, using as an example a typical HALS ferric heme center, [Fe(III)((15)N-coproporphyrin)(CN)2]. The analysis of the experimental g-tensor of [Fe(III)((15)N-coproporphyrin)(CN)2](-) has shown that the widths of the underlying energy distributions for this HALS center are comparable to those found for the rhombic bis-imidazole complex. The greater effect on the g-value distributions for HALS centers is determined by near degeneracy of two of the three lower-energy d-orbitals, d(yz) and d(xz), which contain the unpaired electron.
| S-EPMC3660502 | biostudies-literature