How Single Site Mutations Can Help Understanding Structure Formation of Amyloid β1−40

Amyloid fibrils represent the structural endpoint on the energetic (mis)folding landscape of very many proteins. Physiologically, amyloid fibrils are observed as a characteristic hallmark in misfolding diseases often associated with degenerative and neurodegenerative disorders. In the beginning of the scientific discussion, the focus is laid on the fibrillar state, but over the time it becomes increasingly clear that low molecular weight and transient aggregates are of crucial importance for pathological mechanisms. Structural studies find different intra- and intermolecular contacts for the most well-studied peptide amyloid β (Aβ) depending on the stage of fibrillation. In particular, the contact between residues phenylalanine 19 (F19) and leucine 34 (L34) seems to be highly conserved, suggesting that it must be of particular significance for Aβ misfolding and possibly the pathological properties of the peptide. This review aims to highlight the rational and the usefulness of point mutations in Aβ peptides and their impact on the critical interstrand contact F19−L34 depending on the stage of fibrillation. While the amyloid structure of Aβ is very robust against quite a few modifications, the toxicity of mutated Aβ molecules highly depends on the F19−L34 contact.     

Phase separation of p53 precedes aggregation and is affected by oncogenic mutations and ligands

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF). The mechanism of the formation of the aggregates in the nucleus remains uncertain. The present study demonstrated that the DNA-binding domain of p53 (p53C) underwent phase separation (PS) on the pathway to aggregation under various conditions. p53C phase separated in the presence of the crowding agent polyethylene glycol (PEG). Similarly, mutant p53C (M237I and R249S) underwent PS; however, the process evolved to a solid-like phase transition faster than that in the case of wild-type p53C. The data obtained by microscopy of live cells indicated that transfection of mutant full-length p53 into the cells tended to result in PS and phase transition (PT) in the nuclear compartments, which are likely the cause of the GoF effects. Fluorescence recovery after photobleaching (FRAP) experiments revealed liquid characteristics of the condensates in the nucleus. Mutant p53 tended to undergo gel- and solid-like phase transitions in the nucleus and in nuclear bodies demonstrated by slow and incomplete recovery of fluorescence after photobleaching. Polyanions, such as heparin and RNA, were able to modulate PS and PT in vitro. Heparin apparently stabilized the condensates in a gel-like state, and RNA apparently induced a solid-like state of the protein even in the absence of PEG. Conditions that destabilize p53C into a molten globule conformation also produced liquid droplets in the absence of crowding. The disordered transactivation domain (TAD) modulated both phase separation and amyloid aggregation. In summary, our data provide mechanistic insight into the formation of p53 condensates and conditions that may result in the formation of aggregated structures, such as mutant amyloid oligomers, in cancer. The pathway of mutant p53 from liquid droplets to gel-like and solid-like (amyloid) species may be a suitable target for anticancer therapy.

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF).     

Protein folding through kinetic discrimination

Proteins fold on a micros-ms time scale. However, the number of possible conformations of the polypeptide backbone is so large that random sampling would not allow the protein to fold within the lifetime of the universe, the Levinthal paradox. We show here that a protein chain can fold efficiently with high fidelity if on average native contacts survive longer than non-native ones, that is, if the dissociation rate constant for breakage of a contact is lower for native than for non-native interactions. An important consequence of this finding is that no pathway needs to be specified for a protein to fold. Instead, kinetic discrimination among formed contacts is a sufficient criterion for folding to proceed to the native state. Successful protein folding requires that productive contacts survive long enough to obtain a certain level of probability that other native contacts form before the first interacting unit dissociates. If native contacts survive longer than non-native ones, this prevents misfolding and provides the folding process with directionality toward the native state. If on average all contacts survive equally long, the protein chain is deemed to fold through random search through all possible conformations (i.e., the Levinthal paradox). A modest degree of cooperativity among the native contacts, that is, decreased dissociation rate next to neighboring contacts, shifts the required ratio of dissociation rates into a realistic regime and makes folding a stochastic process with a nucleation step. No kinetic discrimination needs to be invoked in regards to the association process, which is modeled as dependent on the diffusion rate of chain segments.     

Direct measurement of tertiary contact cooperativity in RNA folding

All structured biological macromolecules must overcome the thermodynamic folding problem to populate a unique functional state among a vast ensemble of unfolded and alternate conformations. The exploration of cooperativity in protein folding has helped reveal and distinguish the underlying mechanistic solutions to this folding problem. Analogous dissections of RNA tertiary stability remain elusive, however, despite the central biological importance of folded RNA molecules and the potential to reveal fundamental properties of structured macromolecules via comparisons of protein and RNA folding. We report a direct quantitative measure of tertiary contact cooperativity in a folded RNA. We precisely measured the stability of an independently folding P4-P6 domain from the Tetrahymena thermophila group I intron by single molecule fluorescence resonance energy transfer (smFRET). Using wild-type and mutant RNAs, we found that cooperativity between the two tertiary contacts enhances P4-P6 stability by 3.2 +/- 0.2 kcal/mol.     

Peptide‐binding specificity of the prosurfactant protein C Brichos domain analyzed by electrospray ionization mass spectrometry

The C‐terminal domain of lung surfactant protein C (CTC) precursor (proSP‐C) is involved in folding of the transmembrane segment of proSP‐C. CTC includes a Brichos domain with homologs in cancer‐ and dementia‐associated proteins. Mutations in the Brichos domain cause misfolding of proSP‐C and hence amyloid fibril formation in interstitial lung disease. Electrospray ionization mass spectrometry (ESI‐MS) with collision‐induced dissociation (CID) experiments was applied to study non‐covalent interactions between human recombinant CTC or its Brichos domain, and SP‐C analogs, homotripeptides and peptides designed to model amyloid fibril formation. The results show that the Brichos domain contains the peptide‐binding function of CTC. In titration experiments, apparent dissociation constants (K_D) were in the micromolar range where triple‐valine showed the lowest K_D and triple‐tyrosine the highest. Non‐hydrophobic peptides failed to form complexes with Brichos. CID revealed that complexes with aromatic peptide ligands are more stable in the gas phase than complexes with non‐aromatic ligands. The Brichos domain was also shown to bind fibril‐forming peptides containing aromatic/hydrophobic residues. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrochemical sensing of tumor suppressor protein p53–deoxyribonucleic acid complex stability at an electrified interface

Electrochemical biosensors have the unique ability to convert biological events directly into electrical signals suitable for parallel analysis. Here we utilize specific properties of constant current chronopotentiometric stripping (CPS) in the analysis of protein and DNA–protein complex nanolayers. Rapid potential changes at high negative current intensities (I_str) in CPS are utilized in the analysis of DNA–protein interactions at thiol-modified mercury electrodes. P53 core domain (p53CD) sequence-specific binding to DNA results in a striking decrease in the electrocatalytic signal of free p53. This decrease is related to changes in the accessibility of the electroactive amino acid residues in the p53CD–DNA complex. By adjusting I_str and temperature, weaker non-specific binding can be eliminated or distinguished from the sequence-specific binding. The method also reflects differences in the stabilities of different sequence-specific complexes, including those containing spacers between half-sites of the DNA consensus sequence. The high resolving power of this method is based on the disintegration of the p53CD–DNA complex by the electric field effects at a negatively charged surface and fine adjustment of the millisecond time intervals for which the complex is exposed to these effects. Picomole amounts of p53 proteins and DNA were used for the analysis at full electrode coverage but we show that even 10–20-fold smaller amounts can be analyzed. Our method cannot however take advantage of very low detection limits of the protein CPS detection because low I^_str intensities are deleterious to the p53CD–DNA complex stability at the electrode surface. These data highlight the utility of developing biosensors offering novel approaches for studying real-time macromolecular protein dynamics.     

Selective Inhibition of Aggregation and Toxicity of a Tau‐Derived Peptide using Its Glycosylated Analogues

Protein glycosylation is a ubiquitous post‐translational modification that regulates the folding and function of many proteins. Misfolding of protein monomers and their toxic aggregation are the hallmark of many prevalent diseases. Thus, understanding the role of glycans in protein aggregation is highly important and could contribute both to unraveling the pathology of protein misfolding diseases as well as providing a means for modifying their course for therapeutic purposes. Using β‐O‐linked glycosylated variants of the highly studied Tau‐derived hexapeptide motif VQIVYK, which served as a simplified amyloid model, we demonstrate that amyloid formation and toxicity can be strongly attenuated by a glycan unit, depending on the nature of the glycan itself. Importantly, we show for the first time that not only do glycans hinder self‐aggregation, but the glycosylated peptides are capable of inhibiting aggregation of the non‐modified corresponding amyloid scaffold.     

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid β (1–40)

A small library of rationally designed amyloid β [Aβ(1–40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aβ(1–40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid‐state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aβ(1–40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aβ fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross‐β structure.     

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

The kinase inhibitory domain of the cell cycle regulatory protein p27^Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of ¹³C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.     

Importance of native-state topology for determining the folding rate of two-state proteins

Understanding the relationship between amino acid sequences and folding rate of proteins is a challenging task similar to protein folding problem. In this work, we have analyzed the relative importance of protein sequence and structure for predicting the protein folding rates in terms of amino acid properties and contact distances, respectively. We found that the parameters derived with protein sequence (physical-chemical, energetic, and conformational properties of amino acid residues) show very weak correlation (|r| < 0.39) with folding rates of 28 two-state proteins, indicating that the sequence information alone is not sufficient to understand the folding rates of two-state proteins. However, the maximum positive correlation obtained for the properties, number of medium-range contacts, and alpha-helical tendency reveals the importance of local interactions to initiate protein folding. On the other hand, a remarkable correlation (r varies from -0.74 to -0.88) has been obtained between structural parameters (contact order, long-range order, and total contact distance) and protein folding rates. Further, we found that the secondary structure content and solvent accessibility play a marginal role in determining the folding rates of two-state proteins. Multiple regression analysis carried out with the combination of three properties, beta-strand tendency, enthalpy change, and total contact distance improved the correlation to 0.92 with protein folding rates. The relative importance of existing methods along with multiple-regression model proposed in this work will be discussed. Our results demonstrate that the native-state topology is the major determinant for the folding rates of two-state proteins.     

蛋白质分子的HNP格点模型

从 6 0种球形蛋白质的结构出发 ,采用Miyazawa Jernigan相互作用矩阵 ,计算了蛋白质分子中氨基酸之间的相互作用能 .发现构成蛋白质分子的 2 0种氨基酸可分成疏水 (Hydrophobic ,H)、中性 (Neutral,N)、亲水(Hydrophilic ,P)基团 .在计算它们之间相互作用能的基础上 ,建立了蛋白质分子的HNP格点模型 .用这个模型计算了二维蛋白质分子在自然态 (Nativestate)时的构象性质 .同时研究了氨基酸序列为HHNHNPNHPP HPNPPHPHPPHHPHNH的折叠过程 ,得到其基态能量为 - 6 4 89RT .这能为研究球形蛋白质的构象性质及折叠过程提供一种更合理的格点模型     

Snapshotting the transient conformations and tracing the multiple pathways of single peptide folding using a solid-state nanopore

A fundamental question relating to protein folding/unfolding is the time evolution of the folding of a protein into its precisely defined native structure. The proper identification of transition conformations is essential for accurately describing the dynamic protein folding/unfolding pathways. Owing to the rapid transitions and sub-nm conformation differences involved, the acquisition of the transient conformations and dynamics of proteins is difficult due to limited instrumental resolution. Using the electrochemical confinement effect of a solid-state nanopore, we were able to snapshot the transient conformations and trace the multiple transition pathways of a single peptide inside a nanopore. By combining the results with a Markov chain model, this new single-molecule technique is applied to clarify the transition pathways of the β-hairpin peptide, which shows nonequilibrium fluctuations among several blockage current stages. This method enables the high-throughput investigation of transition pathways experimentally to access previously obscure peptide dynamics, which is significant for understanding the folding/unfolding mechanisms and misfolding of peptides or proteins.

A solid-state nanopore based method is described for resolving protein-folding-related problems via snapshotting the folding intermediates and characterizing the kinetics of a single peptide.     

The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for type-2 diabetes mellitus) was studied by electronic circular dichroism (ECD) and NMR spectroscopy. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return (e.g.; T=37 °C, pH 4.1, c_E5=250 μm , c_NaCl=50 mm , t>4–6 h) that will inevitably gravitate into the amyloid state. The strong B-type far ultraviolet (FUV)-ECD spectra and an unexpectedly strong near ultraviolet (NUV)-ECD signal (Θ_{≈275–285 nm}) indicate that the amyloid phase of E5 is built from monomers of quasi-elongated backbone structure (φ≈−145°, ψ≈+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediates and the buildup of “toxic” early-stage oligomers leading to self-association were identified and monitored as a function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix, exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the β-structured amyloid nucleus, thus the process follows a nucleated growth mechanism.     

Multifunctional 8‐Hydroxyquinoline‐Appended Cyclodextrins as New Inhibitors of Metal‐Induced Protein Aggregation

Mounting evidence suggests a pivotal role of metal imbalances in protein misfolding and amyloid diseases. As such, metal ions represent a promising therapeutic target. In this context, the synthesis of chelators that also contain complementary functionalities to combat the multifactorial nature of neurodegenerative diseases is a highly topical issue. We report two new 8‐hydroxyquinoline‐appended cyclodextrins and highlight their multifunctional properties, including their Cu^II and Zn^II binding abilities, and capacity to act as antioxidants and metal‐induced antiaggregants. In particular, the latter property has been applied in the development of an effective assay that exploits the formation of amyloid fibrils when β‐lactoglobulin A is heated in the presence of metal ions.     

THE DISTRIBUTION FUNCTION OF RESIDUE-RESIDUE CONTACTS IN PROTEIN MOLECULES

In protein molecules each residue has a different ability to form contacts.In this paper,we calculated the number of contacts per residue and investigated the distribution of residue-residue contacts from 495 globular protein molecules using Contacts of Structural Units(CSU)software.It was found that the probability P(n)of amino acid residues having n pairs of contacts in all contacts fits Gaussian distribution very well.The distribution function of residue-residue contacts can be expressed as:P(n)=P_0+aexp[-b(n-n_c)~2].In our calculation,P_0=-0.06,α=11.4,b=-0.04 and n_c=9.0.According to distribution function,we found that those hydrophobic(H)residues including Leu,Val,Ile,Met,Phe,Tyr,Cys,and Trp residues have large values of the most probable number of contact n_c,and hydrophilic(P)residues including Ala,Gly,Thr, His,Glu,Gln,Asp,Asn,Lys,Ser,Arg,and Pro residues have the small ones.We also compare with Fauchere-Pliska hydrophobicity scale(FPH)and the most probable number of contact n_c for 20 amino acid residues,and find that there exists a linear relationship between Fauchere-Pliska hydrophobicity scale(FPH)and the most probable number of contact n_c, and it is expressed as:n_c=a+b×FPH,here α=8.87,and b=1.15.It is important to further explain protein folding and its stability from residue-residue contacts.     

Conformational Dynamics in the Core of Human Y145Stop Prion Protein Amyloid Probed by Relaxation Dispersion NMR

Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using ¹⁵N rotating frame (R_1ρ) relaxation dispersion solid-state nuclear magnetic resonance spectroscopy over a wide range of spin-lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two-state exchange process with a common exchange rate of 1000 s⁻¹, corresponding to protein backbone motion on the timescale of 1 ms, and an excited-state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited-state populations (∼5–15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100–300 μs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.     

Real‐Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

The kinase inhibitory domain of the cell cycle regulatory protein p27^Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D‐DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α‐helical structure, upon binding to cyclin‐dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real‐time observation of ¹³C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time‐dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D‐DNP NMR experiment.     

Capturing intermediate structures of Alzheimer's beta-amyloid, Abeta(1-40), by solid-state NMR spectroscopy

Molecular structures of diffusible amyloid intermediates, commonly observed in misfolding of amyloid proteins into fibrils, have attracted broad interest because the intermediates may be potent neurotoxins responsible for amyloid diseases such as Alzheimer's disease (AD) and because the intermediate structures provide an experimental basis for defining the misfolding pathway. However, owing to the intrinsically unstable and noncrystalline nature of the systems, traditional approaches such as X-ray crystallography and solution NMR have been ineffective for elucidating molecular-level structures of the amyloid intermediates. We present a novel approach using solid-state NMR (SSNMR) that permitted the first site-resolved structural measurement of an intermediate species in fibril formation for a 40-residue Alzheimer's beta-amyloid peptide, Abeta(1-40). In this approach, we combined detection of conformation and morphology changes by fluorescence spectroscopy and electron microscopy and quantitative structural examination for freeze-trapped intermediates by SSNMR. The results provide the initial evidence that a spherical amyloid intermediate of 15-30 nm in diameter exists prior to fibril formation of Abeta(1-40) and that the intermediate involves well-ordered beta-sheets in the C-terminal and hydrophobic core regions. The SSNMR-based approach presented here could be applied to intermediate species of diverse amyloid proteins.     

(α/β)_8桶状蛋白质中紧密接触对的统计性质研究

在计算蛋白质分子中的氨基酸紧密接触对时采用以氨基酸的重心代替Cα 原子的新方法 ,通过计算(α/β) 8桶状蛋白质中的中程和远程紧密接触对的数目 ,研究了不同氨基酸在形成紧密接触对时所具有的不同能力以及在蛋白质结构稳定性中所起的不同作用 .发现在 (α β) 8桶状蛋白质中氨基酸形成的远程紧密接触对数目与它的Fauchere Pliska疏水性实验数值 (FPH)存在着非常好的线性关系 ,而对于氨基酸所形成的中程紧密接触对数目不存在这种关系 .同时还研究了 (α β) 8桶状蛋白质分子大小 ,发现其回转半径大小与表示远程接触对数目的远程次序值LRO存在着关系 .表明这一类 (α β) 8桶状蛋白质具有相似的结构 ,分子的远程接触对数目越多 ,分子越紧密 ,分子尺寸就越小 .同时还研究了不同氨基酸之间形成紧密接触对的能力 .