Slow spontaneous ��-to-�� structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Dynamics of apomyoglobin in the α-to-β transition and of partially unfolded aggregated protein

Changes of molecular dynamics in the α-to-β transition associated with amyloid fibril formation were explored on apomyoglobin (ApoMb) as a model system. Circular dichroism, neutron and X-ray scattering experiments were performed as a function of temperature on the protein, at different solvent conditions. A significant change in molecular dynamics was observed at the α-to-β transition at about 55°C, indicating a more resilient high temperature β structure phase. A similar effect at approximately the same temperature was observed in holo-myoglobin, associated with partial unfolding and protein aggregation. A study in a wide temperature range between 20 and 360 K revealed that a dynamical transition at about 200 K for motions in the 50 ps time scale exists also for a hydrated powder of heat-denatured aggregated ApoMb.     

The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.     

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein,an intrinsically disordered protein

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer. With an increase in the number of –OH groups in polyols except EG, we observe a decrease in lag time for fibrillation at equimolar concentrations, suggesting stronger preferential exclusion of polyols that promotes γ-Syn self-association and oligomerization. The polyols act early during nucleation and their diverse effect on the rate of fibrillation suggests the role of favourable solvent-side chain interactions. With increasing –OH group, polyols stabilize the natively unfolded conformation of γ-Syn under non-fibrillating conditions and delay the structural transition to characteristic β-sheet structure by forming an α-helical intermediate during fibrillation. The results, overall suggest that the effect of osmolytes on IDPs is much more complex than their effect on globular protein stability and aggregation and a fine balance between the dominant unfavourable osmolyte-peptide backbone and favourable osmolyte-charged side chain interactions would govern their stability and aggregation properties.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R₁, R_1ρ, R₂ and η_xy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Are there temperature-dependent structural transitions in the "intrinsically unstructured" protein prothymosin α?

Prothymosin alpha, a typical member of the class of the so-called "intrinsically unstructured" proteins, adopts a random-chain conformation under physiological environmental conditions. An apparent formation of ordered secondary structure and a moderate compaction are observed upon the change from neutral to acid pH at room temperature. We have addressed the question of whether there are temperature-dependent changes of the conformational state of prothymosin alpha at low pH using circular dichroism spectroscopy and static and dynamic light scattering. In contrast to previous investigations, we did not observe a heat-induced conformational transition. For comparison, we have also carried out the same experimental procedures with acid-unfolded phosphoglycerate kinase from yeast. In this case we observed a weak compaction and a slight apparent increase in ordered secondary structure with increasing temperature, probably caused by the higher average hydrophobicity as compared to prothymosin alpha. In the absence of a clear structural transition, we deduce the observed effects result mainly from a progressive redistribution in the population of phi-psi angles of the polypeptide backbone when the temperature is increased. Furthermore, the paper should demonstrate the difficulties in distinguishing between such a progressive change amongst a continuum of states within the ensemble of unfolded conformations from the formation of authentic stable secondary structures in highly unfolded proteins. This problem is not solved presently and convincing evidence can only be supplied by the combination of various experimental techniques.     

Segmental conformational disorder and dynamics in the intrinsically disordered protein α-synuclein and its chain length dependence

Conformational ensembles of fully disordered natural polypeptides represent the starting point of protein refolding initiated by transfer to folding conditions. Thus, understanding the transient properties and dimensions of such peptides under folding conditions is a necessary step in the understanding of their subsequent folding behavior. Such ensembles can also undergo alternative folding and form amyloid structures, which are involved in many neurological degenerative diseases. Here, we performed a structural study of this initial state using time-resolved fluorescence resonance energy transfer analysis of a series of eight partially overlapping double-labeled chain segments of the N-terminal and NAC domains of the α-synuclein molecule. The distributions of end-to-end distance and segmental intramolecular diffusion coefficients were simultaneously determined for eight labeled chain segments. We used the coefficient of variation, C_v, as a measure of the conformational heterogeneity (i.e., structural disorder). With the exception of two segments, the C_vs were characteristic of a fully disordered state of the chain. Subtle deviations from this behavior at the segment labeled in the NAC domain and the segment at the N termini reflected subtle conformational bias that might be related to the initiation of transition to amyloid aggregates. The chain length dependence of the mean segmental end-to-end distance followed a power law as predicted by Flory, but the dependence was steeper than previously predicted, probably due to the contribution of the excluded volume effect, which is more dominant for shorter-chain segments. The observed intramolecular diffusion coefficients (< 10 to ∼ 25 ?²/ns) are only an order of magnitude lower than the common diffusion coefficients of low molecular weight probes. This diffusion coefficient increased with chain length, probably due to the cumulative contributions of minor bond rotations along the chain. These results gave us a reference both for characteristics of a natural unfolded polypeptide at the moment of initiation of folding and for detection of possible initiation sites of the amyloid transition.     

Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc?

A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.     

A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein,an intrinsically disordered protein

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H^N–H^α and H^N–H^N NOEs, values for ³J_HNHα, ¹J_HαCα, ²J_CαN, and ¹J_CαN, as well as chemical shifts of ¹⁵N, ¹³C^α, and ¹³C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PP_II region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the α_R region (10–20%) is found. Analysis of ¹H–¹H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.     

Functional dissection of an intrinsically disordered protein: Understanding the roles of different domains of Knr4 protein in protein�Cprotein interactions

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N‐terminal and large natively unfolded C‐terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two‐hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N‐terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C‐terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.     

Immune cell types involved in early uptake and transport of recombinant mouse prion protein in Peyer’s patches of calves

We have previously reported the early uptake and transport of foreign particles into Peyer’s patches (PPs) of newborn and 2-month-old calves and shown that the peak uptake of particles occurs 6 h after inoculation, in addition to site- and size-related effects on particle uptake. We now report the distribution of immune cells within PPs of the distal ileum in newborn and 2-month-old calves inoculated with carbon black. The types of immune cells involved in the early uptake and transport of recombinant mouse prion protein (rMPrP) within PPs of newborn calf were investigated by using monoclonal antibodies CD11c, CD14, CD68, CD172a, and CD21. CD11c⁺, CD14⁺, CD68⁺, CD172a⁺, and CD21⁺ immune cells were widely distributed in four tissue compartments (villi, dome, interfollicular region, and follicles) of PPs in the distal ileum of newborn and 2-month-old calves, whereas CD11c⁺, CD14⁺, CD172a⁺, and CD21⁺ immune cells were more prominently distributed in the dome areas of newborn calves than in 2-month-old calves. Moreover, CD11c⁺ and CD14⁺ dendritic cells, CD172a⁺ and CD68⁺ macrophages, and CD21⁺ follicular dendritic cells containing rMPrP were primarily observed in the dome and inner follicular regions. The deposition of rMPrP within CD11c⁺, CD14⁺, CD172a⁺, and CD68⁺ cells, but not CD21⁺ cells, was detected in villous regions. rMPrP-positive immune cells within the interfollicular regions included only CD11c⁺ and CD172⁺ cells. Although the particles used in this investigation do not include the infectious prion protein, PrP^Sc, our experimental setup provides a useful model for studying immune cells involved in the early uptake and transport of PrP^Sc.     

Is unphosphorylated Rex,as multifunctional protein of HTLV-1, a fully intrinsically disordered protein? An in silico study

Intracellularlocation of a viral unspliced mRNA in host cell is a crucial factor for normal life of the virus. Rex is a neucleo-cytoplasmic shuffling protein of Human T-cell Leukemia Virus-1(HTLV-1)which has important role in active transport of cargo-containing RNA from nucleus to cytoplasm. Therefore, it plays a crucial role in the disease development by the virus. In spite of its importance, the 3d-structurephosphorylated and unphosphorylated of this protein has not been determined. In this study, first we predicted whether Rex protein is an ordered or disordered protein. In second step protein 3Dstructure of Rex was obtained. The content of disorder-promoting amino acids, flexibility, hydrophobicity, short linear motifs (SLiMs) and protein binding regions and probability of Rex crystallization were calculated by various In Silico methods. The3D models of Rex protein were obtained by various In Silico methods, such as homology modeling, threading and ab initio, including; I-TASSER, LOMETS, SPARSKS, ROBBETA and QUARK servers. By comparing and analyzing Qmean, z-scores and energy levels of selected models, the best structures with highest favored region in Ramachandran plot (higher than 90%) was refined with MODREFINER software. In silico analysis of Rex physicochemical properties and also predicted SLiMs and binding regions sites confirms that unphosphorylated Rex protein in HTLV-1 as Rev protin in HIV is wholly disordered protein belongs to the class of intrinsically disordered proteins with extended disorder (native coils, native pre-molten globules).     

Evidence supporting the role of calpain in the α-processing of amyloid-β precursor protein

Amyloid plaques are a hallmark of the aging and senile dementia brains, yet their mechanism of origins has remained elusive. A central issue is the regulatory mechanism and identity of α-secretase, a protease responsible for α-processing of amyloid-β precursor protein (APP). A remarkable feature of this enzyme is its high sensitivity to a wide range of cellular stimulators, many of which are agonists for Ca(2+) signaling. This feature, together with previous work in our laboratory, has suggested that calpain, a Ca(2+)-dependent protease, plays a key role in APP α-processing. In this study we report that overexpression of the μ-calpain gene in HEK293 cells resulted in a 2.7-fold increase of the protein levels. Measurements of intracellular calpain enzymatic activity revealed that the calpain overexpressing cells displayed a prominent elevation of the activity compared to wild-type cells. When the cells were stimulated by nicotine, glutamate or phorbol 12,13-dibutylester, the activity increase was even more remarkable and sensitive to calpeptin, a calpain inhibitor. Meanwhile, APP secretion from the calpain overexpressing cells was robustly increased under both resting and stimulated conditions over wild-type cells. Furthermore, cell surface biotinylation experiments showed that μ-calpain was clearly detected among the cell surface proteins. These data together support our view that calpain should be a reasonable candidate for α-secretase for further study. This model is discussed with an interesting fact that three other deposited proteins (tau, spectrin and crystalline) are also the known substrates of calpain. Finally we discuss some current misconceptions in senile dementia research.     

Cyclization of the intrinsically disordered α1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.     

N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer

The conformational properties of soluble α-synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N-terminal acetylated α-synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N-terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue-specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N-terminal residues, as well as smaller long-range changes in residues 28-31, 43-46, and 50-66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N-terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated α-synuclein, but that their growth rates are slower. Our results highlight that N-terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular weight species, but does alter the conformational distributions of monomeric α-synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.     

Aggregation/fibrillogenesis of recombinant human prion protein and Gerstmann-Sträussler-Scheinker disease peptides in the presence of metal ions

In this study we investigated the role of Cu(2+), Mn(2+), Zn(2+), and Al(3+) in inducing defective conformational rearrangements of the recombinant human prion protein (hPrP), which trigger aggregation and fibrillogenesis. The research was extended to the fragment of hPrP spanning residues 82-146, which was identified as a major component of the amyloid deposits in the brain of patients affected by Gerstmann-Str?ussler-Scheinker (GSS) disease. Variants of the 82-146 wild-type subunit [PrP-(82-146)(wt)] were also examined, including entirely, [PrP-(82-146)(scr)], and partially scrambled, [PrP-(82-146)(106)(-)(126scr)] and [PrP-(82-146)(127)(-)(146scr)], peptides. Al(3+) strongly stimulated the conversion of native hPrP into the altered conformation, and its potency in inducing aggregation was very high. Despite a lower rate and extent of prion protein conversion into altered isoforms, however, Zn(2+) was more efficient than Al(3+) in promoting organization of hPrP aggregates into well-structured, amyloid-like fibrillar filaments, whereas Mn(2+) delayed and Cu(2+) prevented the process. GSS peptides underwent the fibrillogenesis process much faster than the full-length protein. The intrinsic ability of PrP-(82-146)(wt) to form fibrillar aggregates was exalted in the presence of Zn(2+) and, to a lesser extent, of Al(3+), whereas Cu(2+) and Mn(2+) inhibited the conversion of the peptide into amyloid fibrils. Amino acid substitution in the neurotoxic core (sequence 106-126) of the 82-146 fragment reduced its amyloidogenic potential. In this case, the stimulatory effect of Zn(2+) was lower as compared to the wild-type peptide; on the contrary Al(3+) and Mn(2+) induced a higher propensity to fibrillation, which was ascribed to different binding modalities to GSS peptides. In all cases, alteration of the 127-146 sequence strongly inhibited the fibrillogenesis process, thus suggesting that integrity of the C-terminal region was essential both to confer amyloidogenic properties on GSS peptides and to activate the stimulatory potential of the metal ions.     

Characterization and strain distribution of four alleles at the hemoglobin α-chain structural locus in the mouse

In a survey of mice from 40 inbred strains, largely previously untested, four alleles were distinguished at the Hba locus, determining structure of adult mouse hemoglobin chain. This finding supports and extends previous sequence studies by others. Methods are given by which each phenotype was characterized by its solubility profile at varying pH and by its chromatography pattern. Concordance was complete between histidine-positive T-4 (defining Hba ^c) and high-intermediate solubility profile. In three inbred strains, a distinct new low-solubility profile, not associated with his-positive T-4, was recognized, and mice with this phenotype were classified as Hba ^d. Implications of observed widely distributed four-allele polymorphism of mouse hemoglobin -chain structure are discussed.This investigation was supported in part by NIH research grant CA-01074 from the National Cancer Institute and in part by the Virginia and D. K. Ludwig Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

The role of protein–solvent hydrogen bond dynamics in the structural relaxation of a protein in glycerol versus water

We used MD simulations to investigate the dependence of the dynamics of a soluble protein, RNase A, on temperature and solvent environment. Consistent with neutron scattering data, the simulations predict that the protein undergoes a dynamical transition in both glycerol and aqueous solutions that is absent in the dry protein. The temperature of the transition is higher, while the rate of increase with temperature of the amplitudes of motion on the 100 ps timescale is lower, in glycerol versus water. Analysis of the dynamics of hydrogen bonds revealed that the protein dynamical transition is connected to the relaxation of the protein-solvent hydrogen bond network, which, in turn, is associated with solvent translational diffusion. Thus, it appears that the role of solvent dynamics in affecting the protein dynamical transition is qualitatively similar in water and glycerol.