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The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (15 February 2023) | Viewed by 13647

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Prof. Dr. Tomohiro Mizobata

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Guest Editor

Faculty of Engineering, Chemistry and Biotechnology, Tottori University, Tottori 683-8503, Japan
Interests: protein science; protein folding; molecular chaperones

Special Issue Information

Dear Colleagues,

Recent advances in protein structural dynamics in vitro and in vivo have revealed the richness of the protein structural landscape. In order to achieve biological activity, proteins utilize many diverse states, up to and including the unfolded and aggregated states. Moreover, proteins transition between these states dynamically in response to numerous stimuli and at times recruit the help of various chaperone proteins to attain this goal.

In this issue, we search for examples of protein structural dynamism that lead to diverse biological activities. Such examples include the action and effects of molecular chaperones on protein structure and activity, the accumulation and control of fibrillar protein deposits that sometimes lead to unique and complex functions, and the maintenance and utilization of intrinsically unfolded proteins and sequence regions that allow a diversity of cellular function and modulation.

Prof. Dr. Tomohiro Mizobata
Guest Editor

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Keywords

intrinsically unfolded proteins and regions
molecular chaperones
amyloid fibrils
functional amyloids
proteostasis

Published Papers (7 papers)

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Research

17 pages, 2374 KiB

Open AccessArticle

Protein Unfolding—Thermodynamic Perspectives and Unfolding Models

by Joachim Seelig and Anna Seelig

Int. J. Mol. Sci. 2023, 24(6), 5457; https://doi.org/10.3390/ijms24065457 - 13 Mar 2023

Cited by 2 | Viewed by 1715

Abstract

We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change C_p(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile C_p(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical–mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical–mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical–mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies. Full article

(This article belongs to the Special Issue The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils)

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16 pages, 3741 KiB

Open AccessArticle

The Regulatory Mechanism of Transthyretin Irreversible Aggregation through Liquid-to-Solid Phase Transition

by Guangfei Duan, Yanqin Li, Meimei Ye, Hexin Liu, Ning Wang and Shizhong Luo

Int. J. Mol. Sci. 2023, 24(4), 3729; https://doi.org/10.3390/ijms24043729 - 13 Feb 2023

Viewed by 1534

Abstract

Transthyretin (TTR) aggregation and amyloid formation are associated with several ATTR diseases, such as senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). However, the mechanism that triggers the initial pathologic aggregation process of TTR remains largely elusive. Lately, increasing evidence has suggested that many proteins associated with neurodegenerative diseases undergo liquid–liquid phase separation (LLPS) and subsequent liquid-to-solid phase transition before the formation of amyloid fibrils. Here, we demonstrate that electrostatic interactions mediate LLPS of TTR, followed by a liquid-solid phase transition, and eventually the formation of amyloid fibrils under a mildly acidic pH in vitro. Furthermore, pathogenic mutations (V30M, R34T, and K35T) of TTR and heparin promote the process of phase transition and facilitate the formation of fibrillar aggregates. In addition, S-cysteinylation, which is a kind of post-translational modification of TTR, reduces the kinetic stability of TTR and increases the propensity for aggregation, while another modification, S-sulfonation, stabilizes the TTR tetramer and reduces the aggregation rate. Once TTR was S-cysteinylated or S-sulfonated, they dramatically underwent the process of phase transition, providing a foundation for post-translational modifications that could modulate TTR LLPS in the context of pathological interactions. These novel findings reveal molecular insights into the mechanism of TTR from initial LLPS and subsequent liquid-to-solid phase transition to amyloid fibrils, providing a new dimension for ATTR therapy. Full article

(This article belongs to the Special Issue The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils)

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22 pages, 2208 KiB

Open AccessArticle

Effects of Molecular Crowding and Betaine on HSPB5 Interactions, with Target Proteins Differing in the Quaternary Structure and Aggregation Mechanism

by Vera A. Borzova, Svetlana G. Roman, Anastasiya V. Pivovarova and Natalia A. Chebotareva

Int. J. Mol. Sci. 2022, 23(23), 15392; https://doi.org/10.3390/ijms232315392 - 06 Dec 2022

Cited by 1 | Viewed by 1116

Abstract

The aggregation of intracellular proteins may be enhanced under stress. The expression of heat-shock proteins (HSPs) and the accumulation of osmolytes are among the cellular protective mechanisms in these conditions. In addition, one should remember that the cell environment is highly crowded. The antiaggregation activity of HSPB5 and the effect on it of either a crowding agent (polyethylene glycol (PEG)) or an osmolyte (betaine), or their mixture, were tested on the aggregation of two target proteins that differ in the order of aggregation with respect to the protein: thermal aggregation of glutamate dehydrogenase and DTT-induced aggregation of lysozyme. The kinetic analysis of the dynamic light-scattering data indicates that crowding can decrease the chaperone-like activity of HSPB5. Nonetheless, the analytical ultracentrifugation shows the protective effect of HSPB5, which retains protein aggregates in a soluble state. Overall, various additives may either improve or impair the antiaggregation activity of HSPB5 against different protein targets. The mixed crowding arising from the presence of PEG and 1 M betaine demonstrates an extraordinary effect on the oligomeric state of protein aggregates. The shift in the equilibrium of HSPB5 dynamic ensembles allows for the regulation of its antiaggregation activity. Crowding can modulate HSPB5 activity by affecting protein–protein interactions. Full article

(This article belongs to the Special Issue The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils)

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18 pages, 5069 KiB

Open AccessArticle

Formation of Fibrils by the Periplasmic Molecular Chaperone HdeB from Escherichia coli

by Yui Nakata, Yuuto Kitazaki, Hitomi Kanaoka, Erika Shingen, Rina Uehara, Kunihiro Hongo, Yasushi Kawata and Tomohiro Mizobata

Int. J. Mol. Sci. 2022, 23(21), 13243; https://doi.org/10.3390/ijms232113243 - 31 Oct 2022

Viewed by 1308

Abstract

The molecular chaperones HdeA and HdeB of the Escherichia coli (E. coli) periplasm protect client proteins from acid denaturation through a unique mechanism that utilizes their acid denatured states to bind clients. We previously demonstrated that the active, acid-denatured form of HdeA is also prone to forming inactive, amyloid fibril-like aggregates in a pH-dependent, reversible manner. In this study, we report that HdeB also displays a similar tendency to form fibrils at low pH. HdeB fibrils were observed at pH < 3 in the presence of NaCl. Similar to HdeA, HdeB fibrils could be resolubilized by a simple shift to neutral pH. In the case of HdeB, however, we found that after extended incubation at low pH, HdeB fibrils were converted into a form that could not resolubilize at pH 7. Fresh fibrils seeded from these “transformed” fibrils were also incapable of resolubilizing at pH 7, suggesting that the transition from reversible to irreversible fibrils involved a specific conformational change that was transmissible through fibril seeds. Analyses of fibril secondary structure indicated that HdeB fibrils retained significant alpha helical content regardless of the conditions under which fibrils were formed. Fibrils that were formed from HdeB that had been treated to remove its intrinsic disulfide bond also were incapable of resolubilizing at pH 7, suggesting that certain residual structures that are retained in acid-denatured HdeB are important for this protein to recover its soluble state from the fibril form. Full article

(This article belongs to the Special Issue The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils)

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12 pages, 3041 KiB

Open AccessArticle

The Disordered EZH2 Loop: Atomic Level Characterization by ¹H^N- and ¹H^α-Detected NMR Approaches, Interaction with the Long Noncoding HOTAIR RNA

by Csenge Lilla Szabó, Beáta Szabó, Fanni Sebák, Wolfgang Bermel, Agnes Tantos and Andrea Bodor

Int. J. Mol. Sci. 2022, 23(11), 6150; https://doi.org/10.3390/ijms23116150 - 30 May 2022

Cited by 4 | Viewed by 1910

Abstract

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the ¹H^N-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, ¹H^α-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407–Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2^T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR₁₄₀ (1–140 nt) highlight that the Thr367–Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein–RNA interaction as a “fuzzy” complex. Full article

(This article belongs to the Special Issue The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils)

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20 pages, 5855 KiB

Open AccessArticle

Entropy-Enthalpy Compensations Fold Proteins in Precise Ways

by Jiacheng Li, Chengyu Hou, Xiaoliang Ma, Shuai Guo, Hongchi Zhang, Liping Shi, Chenchen Liao, Bing Zheng, Lin Ye, Lin Yang and Xiaodong He

Int. J. Mol. Sci. 2021, 22(17), 9653; https://doi.org/10.3390/ijms22179653 - 06 Sep 2021

Cited by 10 | Viewed by 3377

Abstract

Exploring the protein-folding problem has been a longstanding challenge in molecular biology and biophysics. Intramolecular hydrogen (H)-bonds play an extremely important role in stabilizing protein structures. To form these intramolecular H-bonds, nascent unfolded polypeptide chains need to escape from hydrogen bonding with surrounding polar water molecules under the solution conditions that require entropy-enthalpy compensations, according to the Gibbs free energy equation and the change in enthalpy. Here, by analyzing the spatial layout of the side-chains of amino acid residues in experimentally determined protein structures, we reveal a protein-folding mechanism based on the entropy-enthalpy compensations that initially driven by laterally hydrophobic collapse among the side-chains of adjacent residues in the sequences of unfolded protein chains. This hydrophobic collapse promotes the formation of the H-bonds within the polypeptide backbone structures through the entropy-enthalpy compensation mechanism, enabling secondary structures and tertiary structures to fold reproducibly following explicit physical folding codes and forces. The temperature dependence of protein folding is thus attributed to the environment dependence of the conformational Gibbs free energy equation. The folding codes and forces in the amino acid sequence that dictate the formation of β-strands and α-helices can be deciphered with great accuracy through evaluation of the hydrophobic interactions among neighboring side-chains of an unfolded polypeptide from a β-strand-like thermodynamic metastable state. The folding of protein quaternary structures is found to be guided by the entropy-enthalpy compensations in between the docking sites of protein subunits according to the Gibbs free energy equation that is verified by bioinformatics analyses of a dozen structures of dimers. Protein folding is therefore guided by multistage entropy-enthalpy compensations of the system of polypeptide chains and water molecules under the solution conditions. Full article

(This article belongs to the Special Issue The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils)

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13 pages, 2319 KiB

Open AccessArticle

Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts

by Thomas Näsström, Tobias Dahlberg, Dmitry Malyshev, Jörgen Ådén, Per Ola Andersson, Magnus Andersson and Björn C. G. Karlsson

Int. J. Mol. Sci. 2021, 22(17), 9334; https://doi.org/10.3390/ijms22179334 - 28 Aug 2021

Cited by 3 | Viewed by 1752

Abstract

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs. Full article

(This article belongs to the Special Issue The Multiple and Dynamic Faces of Protein Structure: Folded States, Intrinsically Unfolded States, and Aggregates/Fibrils)

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