The role of the unfolded state in hairpin stability

The effects of a T3S mutation on the stability of a 3:5 beta-hairpin forming peptide (YITNSNGTWT) are investigated. Molecular dynamics simulations in explicit water indicate that the wild-type peptide forms a stable hairpin whereas the T3S mutant does not, in agreement with the experimental data. Thermodynamic integration calculations for the mutation of Thr to Ser suggest that the free-energy changes in the folded state are small, but the corresponding changes in the unfolded state are large and favorable. One of the main reasons for the difference appears to be the formation of a stable cluster involving the Tyr1 and Ser3 hydroxyl groups and their interaction with the C-terminal carboxylate group, which was observed after unfolding of the T3S mutant. Further analysis of the side-chain preferences of Thr and Ser indicate that the corresponding cluster in the wild-type peptide is unstable due to the high preference of the Thr chi1 dihedral for g+ states, which appeared to be incompatible with formation of a stable cluster. The results suggest that one should consider the nature of the unfolded state when attempting to fully explain the effects of mutations on hairpin stability.     

Inverse electrostatic effect: electrostatic repulsion in the unfolded state stabilizes a leucine zipper

The pH-dependent stability of a protein is strongly affected by electrostatic interactions between ionizable residues in the folded as well as unfolded state. Here we characterize the individual contributions of charged Glu and His residues to stability and determine the NMR structure of the designed, heterodimeric leucine zipper AB consisting of an acidic A chain and a basic B chain. Thermodynamic parameters are compared with those of the homologous leucine zipper AB(SS) in which the A and B chains are disulfide-linked. NMR structures of AB based on (1)H NMR data collected at 600 MHz converge, and formation of the same six interchain salt bridges found previously in disulfide-linked AB(SS) [Marti, D. N., and Bosshard, H. R. (2003) J. Mol. Biol. 330, 621-637] is indicated. While the structures of AB and AB(SS) are very similar, their pH-dependent relative stabilities are strikingly different. The stability of AB peaks at pH approximately 4.5 and is higher at pH 8 than at pH 2. In contrast, AB(SS) is most stable at acidic pH where no interhelical salt bridges are formed. The different energetic contributions of charged Glu and His residues to stability of the two coiled coil structures were evaluated from pK(a) shifts induced by folding. The six charged Glu residues involved in salt bridges stabilize leucine zipper AB by 4.5 kJ/mol yet destabilize disulfide-linked AB(SS) by -1.1 kJ/mol. Two non-ion-paired Glu charges destabilize AB by only -1.8 kJ/mol but AB(SS) by -5.6 kJ/mol. The higher relative stability of AB at neutral pH is not caused by more favorable electrostatic interactions in the folded leucine zipper. It is due mainly to unfavorable electrostatic interactions in the unfolded A and B chains and may therefore be called an inverse electrostatic effect. This study illustrates the importance of residual interactions in the unfolded state and how the energetics of the unfolded state affect the stability of the folded protein.     

Phosphorylation modulates the alpha-helical structure and polymerization of a peptide from the third tau microtubule-binding repeat

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of alpha-helices [Biochemistry (2002) 41, 7150-5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at approximately 1665 and approximately 1675 cm(-1), which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.     

The self-assembly ability of the first microtubule-binding repeat from tau and its modulation by phosphorylation

Aggregation of abnormally phosphorylated tau in the form of tangs of paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease (AD) and other tauopathies. It is of fundamental importance to study the mechanism of PHF formation and its modulation by phosphorylation. In this work, we have focused on the first microtubule-binding repeat of tau encompassing an abnormal phosphorylation site Ser262. The assembly propensities of this repeat and its corresponding phosphorylated form were investigated by turbidity and electron microscopy. Additionally, conformation of the two peptides is also analyzed through circular dichroism (CD) and NMR spectroscopy. Our results reveal that both of them are capable of self-assembly and phosphorylation at Ser262 could speed up the process of assembly. A possible mechanism of PHF formation is proposed and enhancing effect of phosphorylation on assembly provides an explanation to its toxicity in Alzheimer's disease.     

Phosphorylation modulates the local conformation and self-aggregation ability of a peptide from the fourth tau microtubule-binding repeat

Phosphorylation of tau protein modulates both its physiological role and its aggregation into paired helical fragments, as observed in Alzheimer's diseased neurons. It is of fundamental importance to study paired helical fragment formation and its modulation by phosphorylation. This study focused on the fourth microtubule-binding repeat of tau, encompassing an abnormal phosphorylation site, Ser356. The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investigated using turbidity, thioflavin T fluorescence and electron microscopy. There is evidence for a conformational change in the fourth microtubule-binding repeat of tau peptide upon phosphorylation, as well as changes in aggregation activity. Although both tau peptides have the ability to aggregate, this is weaker in the phosphorylated peptide. This study reveals that both tau peptides are capable of self-aggregation and that phosphorylation at Ser356 can modulate this process.     

Biological effect of a novel mutation in the third leucine-rich repeat of human luteinizing hormone receptor

A novel heterozygous mutation A340T leading to the substitution of Phe for the conserved amino acid Ile114 was identified by nucleotide sequencing of the human LH/chorionic gonadotropin receptor (hLHR) of a patient with Leydig cell hypoplasia. This mutation is located in the third leucine-rich repeat in the ectodomain of the hLHR. In vitro expression studies demonstrated that this mutation results in reduced ligand binding and signal transduction of the receptor. Studies of hLHR constructs in which various amino acids were substituted for the conserved Ile114 showed that receptor activity is sensitive to changes in size, shape, and charge of the side chain. A homology model of the wild-type hLHR ectodomain was made, illustrating the packing of conserved hydrophobic side chains in the protein core. Substitution of Ile114 by Phe might disrupt intermolecular contacts between hormone and receptor. This mutation might also affect an LHR-dimer interaction. Thus, the I114F mutation reduces ligand binding and signal transduction by the hLHR, and it is partially responsible for Leydig cell hypoplasia in the patient.     

The contribution of electrostatics to hydrogen exchange in the unfolded protein state

Although electrostatics have long been recognized to play an important role in hydrogen exchange (HX) with solvent, the quantitative assessment of its magnitude in the unfolded state has hitherto been lacking. This limits the utility of HX as a quantitative method to study protein stability, folding, and dynamics. Using the intrinsically disordered human protein α-synuclein as a proxy for the unfolded state, we show that a hybrid mean-field approach can effectively compute the electrostatic potential at all backbone amide positions along the chain. From the electrochemical potential, a fourfold reduction in hydroxide concentration near the protein backbone is predicted for the C-terminal domain, a prognosis that is in direct agreement with experimentally derived protection factors from NMR spectroscopy. Thus, impeded HX for the C-terminal region of α-synuclein is not the result of intramolecular hydrogen bonding and/or structure formation.     

Possible role of each repeat structure of the microtubule-binding domain of the tau protein in in vitro aggregation

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1-R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by (1)H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1-R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed.     

The unfolded state of NTL9 is compact in the absence of denaturant

Interest in the unfolded state of proteins has grown with the realization that this state can have considerable structure in the absence of denaturants. Natively unfolded proteins, mutations that unfold proteins under native conditions, and changes in pH that induce unfolding are attractive models for the unfolded state in the absence of denaturant. The unfolded state of the N-terminal domain of ribosomal protein L9 (NTL9) was previously shown to contain significant non-native electrostatic interactions [Cho, J. H., Sato, S., and Raleigh, D. P. (2004) J. Mol. Biol. 338, 827-837]. NTL9 has a mixed alpha-beta structure and folds via a two-state mechanism. We have generated a model of the unfolded state of NTL9 in the absence of denaturant by substitution of an alanine for phenylalanine 5 located in the core of this protein. The CD spectrum of the variant, denoted as F5A, exhibits significantly less structure than the wild type; however, the mean residue ellipticity of F5A at 222 nm (-8200 deg cm(2) dmol(-)(1)) is considerably larger than expected for a fully unfolded protein, indicating that residual secondary structure is populated. F5A also has more residual structure than the urea-unfolded wild type. The stability of F5A is estimated to be at least 1 kcal/mol unfavorable, showing that the unfolded state is populated to 84% or more. NMR pulsed-field gradient measurements yield a hydrodynamic radius of 16.1 A for wild-type NTL9 and 20.8 A for the F5A variant in native buffer. The physiologically relevant unfolded state of wild-type NTL9 is likely to be even more compact than F5A since the mutation should reduce the level of hydrophobic clustering in the unfolded state in the absence of denaturant. The hydrodynamic radius of F5A increases to 25.9 A in 8 M urea, and a value of 23.5 A is obtained for the wild type under similar conditions. The results show that the unfolded state of F5A in the absence of denaturant is more compact and contains more structure than the urea-unfolded form.     

Toward resolution of ambiguity for the unfolded state

The unfolded states in proteins and nucleic acids remain weakly understood despite their importance in folding processes; misfolding diseases (Parkinson's and Alzheimer's); natively unfolded proteins (as many as 30% of eukaryotic proteins, according to Fink); and the study of ribozymes. Research has been hindered by the inability to quantify the residual (native) structure present in an unfolded protein or nucleic acid. Here, a scaling model is proposed to quantify the molar degree of folding and the unfolded state. The model takes a global view of protein structure and can be applied to a number of analytic methods and to simulations. Three examples are given of application to small-angle scattering from pressure-induced unfolding of SNase, from acid-unfolded cytochrome c, and from folding of Azoarcus ribozyme. These examples quantitatively show three characteristic unfolded states for proteins, the statistical nature of a protein folding pathway, and the relationship between extent of folding and chain size during folding for charge-driven folding in RNA.     

Marked difference between self-aggregations of first and fourth repeat peptides on tau microtubule-binding domain in acidic solution

The heparin-induced self-aggregation behaviours of four repeat peptides (R1-R4) in an acidic solution (pH = 4.5) were investigated by fluorescence and circular dichroism (CD) measurements and compared with those in a neutral solution (pH = 7.5). In contrast with the self-aggregation-resistive behaviours of the R1 and R4 repeat peptides in the neutral solution, the R4 peptide formed a filament similarly to the R2 and R3 peptides in the acidic solution, whereas the R1 peptide still showed resistive behaviour for filament formation. This is the first report on the markedly different self-aggregation behaviours of the first and fourth repeat peptides on tau microtubule-binding domain.     

Helix formation and the unfolded state of a 52-residue helical protein

A growing class of proteins in biological processes has been found to be unfolded on isolation under normal solution conditions. We have used NMR spectroscopy to characterize the structural and dynamic properties of the unfolded and partially folded states of a 52-residue alanine-rich protein (Ala-14) at temperatures from -5 degrees C to 40 degrees C. At 40 degrees C, alanine residues in Ala-14 adopt phi and psi angles, consistent with a significant ensemble population of polyproline II conformation. Analysis of relaxation rates in the protein reveals that a series of residues, Gln 35-Ala 36-Ala 37-Lys 38-Asp 39-Asp 40-Ala 41-Ala 42, displays slow motional dynamics at both -5 degrees C and 40 degrees C. Temperature-dependent chemical shift changes indicate that this region is the site of helix initiation. The remaining N-terminal residues become increasingly dynamic as they extend from the nucleation site. The C terminus remains dynamic and changes less with temperature, indicating it is relatively unstructured. Ala-14 provides a high-resolution portrait of the unfolded state and the process of helix nucleation and propagation in the absence of tertiary contacts, information that bears on early events in protein folding.     

Truncated Tau with the Fyn-binding domain and without the microtubule-binding domain hinders the myelinating capacity of an oligodendrocyte cell line

The mechanisms underlying developmental myelination have therapeutic potential following CNS injury and degeneration. We report that transplanted central glial (CG)-4 cells had a diminished myelinating capacity in myelin-deficient (md) rats when cells express a mutated form of Tau (Tau [688]), which binds Fyn but not the microtubules. In the brain of the md rats, Tau [688]-transfected CG-4 cells displayed a decrease in cellular process outgrowth and myelination; in the spinal cord the extent of myelination rostral and caudal to the injection site was decreased. In contrast, control Tau [605]-transfected CG-4 cells formed long cellular processes and substantial areas of myelin both in the brain and spinal cord. In culture, Tau [688]-transfected CG-4 cells displayed a decrease in cellular process outgrowth, and Fyn localized largely in the cell body, not the processes. Thus, Tau in oligodendrocytes plays a key role in myelination, and a functional Tau-Fyn interaction might have therapeutic potential during demyelination and myelin repair following CNS injury and degeneration.     

Phosphorylation regulates fibrillation of an aggregation core peptide in the second repeat of microtubule-binding domain of human tau

Hyperphosphorylation of the microtubule-associated protein tau is believed to play a crucial role in the neurofibrillary tangles formation in Alzheimer’s disease brain. In this study, fibril formation of peptides containing the critical sequences for tau aggregation VQIINK and a plausible serine phosphorylation site of tau at its C-terminal was investigated. All the peptides formed fibrils with the typical cross-β structural core. However, stability of the fibrils was highly sensitive to the pH conditions for the phosphorylated VQIINK peptide, suggesting a regulatory role of phosphorylation for the amyloid-formation of tau.     

Linkage-dependent contribution of repeat peptides to self-aggregation of three- or four-repeat microtubule-binding domains in tau protein

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism for the tau protein, it is still unclear how it is transformed from a normal structure in a neuron. To examine the linkage-dependent contribution of each repeat peptide (R1-R4) to filament formation of the three- or four-repeat microtubule-binding domain (MBD) in the tau protein, four two-repeat peptides (R12, R13, R23 and R34) and two three-repeat peptides (R123 and R234) were prepared, and their in vitro self-aggregation was investigated by thioflavin S fluorescence and circular dichroism measurements, and by electron microscopy in neutral buffer (pH 7.6). Comparison of these aggregation behaviors with previous results for single-repeat peptides and wild-type 3RMBD (R134) and 4RMBD (R1234) indicated that (a) the two-repeat R23, not the R2 or R3 single repeat, forms the core structure in self-aggregation of 4RMBD, whereas that of 3RMBD comprises the R3 single repeat, (b) co-existence of R1 and R4 repeats is necessary for the aggregation behavior inherent in 3RMBD and 4RMBD, whereas the R1 or R4 repeat alone functions as a repressor or modifier of the filament formation, (c) 4RMBD aggregation is accompanied by R1-driven transition from random and alpha-helix structures to a beta-sheet structure, whereas 3RMBD aggregation involves three-repeat R134-specific transition from a random structure to an alpha-helix structure without the participation of a beta-sheet structure, and (d) the peptides that include the R1 repeat form a long filament irrespective of the absence or presence of the R4 repeat, whereas those that include the R4 repeat, but not the R1 repeat, form a relatively short filament. To the best of our knowledge, a systematic study of the linkage-dependent contribution of each repeat peptide to the paired helical filament formation of tau MBD has not been carried out previously, and thus the present information is useful for understanding the essence of the filament formation of tau MBD.     

The quantum mechanically mixed-spin state in a non-symbiotic plant hemoglobin: the effect of distal mutation on AHb1 from Arabidopsis thaliana

Non-symbiotic hemoglobins are hexacoordinated heme proteins found in all plants. To gain insight into the importance of the heme hexacoordination and the coordinated distal histidine in general for the possible physiological functions of these proteins, the distal His(E7) of Arabidopsis thaliana hemoglobin (AHb1) was substituted by a leucine residue. The heme properties of the wild-type and mutant proteins have been characterized by electronic absorption, resonance Raman and electron paramagnetic resonance spectroscopic studies at room and low temperatures. Significant differences between the wild-type and mutant proteins have been detected. The most striking is the formation of an uncommon quantum mechanically mixed-spin heme species in the mutant. This is the first observation of such a spin state in a plant hemoglobin. The proportion of this species, which at room temperature coexists with a minor pentacoordinated high-spin form, increases markedly at low temperature.     

Characterization of the unfolded state of bovine alpha-lactalbumin and comparison with unfolded states of homologous proteins

The unfolded states of three homologous proteins with a very similar fold have been investigated by heteronuclear NMR spectroscopy. Secondary structure propensities as derived from interpretation of chemical shifts and motional restrictions as evidenced by heteronuclear (15)N relaxation rates have been analyzed in the reduced unfolded states of hen lysozyme and the calcium-binding proteins bovine alpha-lactalbumin and human alpha-lactalbumin. For all three proteins, significant deviations from random-coil predictions can be identified; in addition, the unfolded states also differ from each other, despite the fact that they possess very similar structures in their native states. Deviations from random-coil motional properties are observed in the alpha- and the beta-domain in bovine alpha-lactalbumin and lysozyme, while only regions within the alpha-domain deviate in human alpha-lactalbumin. The motional restrictions and residual secondary structure are determined both by the amino acid sequence of the protein and by residual long-range interactions. Even a conservative single point mutation from I to L in a highly conserved region between the two alpha-lactalbumins results in considerable differences in the motional properties. Given the differences in oxidative folding between hen lysozyme and alpha-lactalbumin, the results obtained on the unfolded states suggest that residual long-range interactions, i.e., those between the alpha- and the beta-domain of lysozyme, may act as nucleation sites for protein folding, while this property of residual structure is replaced by the calcium-binding site between the domains in alpha-lactalbumin.