-
Protein Science : a Publication of the... Jun 2024The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets....
The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets. Their size, ease of expression, and apparent high stability makes them excellent candidates for a new class of protein drugs. However, beyond circular dichroism melts and hydrogen/deuterium exchange experiments, little is known about their dynamics, especially at the elevated temperatures they seemingly tolerate quite well. To address that and gain insight for future designs, we have focused on identifying unintended and previously overlooked heat-induced structural and chemical changes in a particularly stable model miniprotein, EHEE_rd2_0005. Nuclear magnetic resonance (NMR) studies suggest the presence of dynamics on multiple time and temperature scales. Transiently elevating the temperature results in spontaneous chemical deamidation visible in the NMR spectra, which we validate using both capillary electrophoresis and mass spectrometry (MS) experiments. High temperatures also result in greatly accelerated intrinsic rates of hydrogen exchange and signal loss in NMR heteronuclear single quantum coherence spectra from local unfolding. These losses are in excellent agreement with both room temperature hydrogen exchange experiments and hydrogen bond disruption in replica exchange molecular dynamics simulations. Our analysis reveals important principles for future miniprotein designs and the potential for high stability to result in long-lived alternate conformational states.
Topics: Hot Temperature; Nuclear Magnetic Resonance, Biomolecular; Molecular Dynamics Simulation; Protein Conformation; Proteins; Protein Stability
PubMed: 38757381
DOI: 10.1002/pro.4991 -
Proteins May 2024FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD)....
FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD). This domain can exist as a monomer or a domain swapped dimer. In addition to the FHD, the leucine zipper region (LZ) of FOXP2 is also believed to be associated with both DNA binding and oligomerization. To better understand the relationship between DNA binding and oligomerization of FOXP2, we investigated its structure, stability and dynamics, focusing specifically on the FHD and the LZ. We did this by using two constructs: one containing the isolated FHD and one containing both the LZ and the FHD (LZ-END). We demonstrate in this work, that while the FHD maintains a monomeric form that is capable of binding DNA, the LZ-END undergoes a dynamic transition between oligomeric states in the presence of DNA. Our findings suggest that FOXP2's LZ domain influences DNA binding affinity through a change in oligomeric state. We show through hydrogen exchange mass spectroscopy that certain parts of the FHD and interlinking region become less dynamic when in the presence of DNA, confirming DNA binding and oligomerization in these regions. Moreover, the detection of a stable equilibrium intermediate state during LZ-END unfolding supports the idea of cooperation between these two domains. Overall, our study sheds light on the interplay between two FOXP2 domains, providing insight into the protein's ability to respond dynamically to DNA, and enriching our understanding of FOXP2's role in gene regulation.
PubMed: 38747678
DOI: 10.1002/prot.26699 -
Protein Science : a Publication of the... Jun 2024Differential scanning fluorimetry (DSF) is a method to determine the apparent melting temperature (Tma) of a purified protein. In DSF, the raw unfolding curves from...
Differential scanning fluorimetry (DSF) is a method to determine the apparent melting temperature (Tma) of a purified protein. In DSF, the raw unfolding curves from which Tma is calculated vary widely in shape and complexity. However, the tools available for calculating Tma are only compatible with the simplest of DSF curves, hindering many otherwise straightforward applications of the technology. To overcome this limitation, we designed new mathematical models for Tma calculation that accommodate common forms of variation in DSF curves, including the number of transitions, the presence of high initial signal, and temperature-dependent signal decay. When tested these models against DSFbase, an open-source database of 6235 raw, real-life DSF curves, these models outperformed the existing standard approaches of sigmoid fitting and maximum of the first derivative. To make these models accessible, we created an open-source software and website, DSFworld (https://gestwickilab.shinyapps.io/dsfworld/). In addition to these improved fitting capabilities, DSFworld also includes features that overcome the practical limitations of many analysis workflows, including automatic reformatting of raw data exported from common qPCR instruments, labeling of data based on experimental variables, and flexible interactive plotting. We hope that DSFworld will enable more streamlined and accurate calculation of Tma values for DSF experiments.
Topics: Fluorometry; Software; Transition Temperature; Proteins
PubMed: 38747440
DOI: 10.1002/pro.5022 -
Protein Science : a Publication of the... Jun 2024Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological...
Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.
Topics: Calorimetry, Differential Scanning; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Protein Domains; Protein Stability; Wheat Germ Agglutinins
PubMed: 38747397
DOI: 10.1002/pro.5020 -
Research Square Apr 2024The homotetrameric thermosensitive transient receptor potential vanilloid 1-4 (TRPV1-4) channels in sensory neurons are strongly responsive to heat stimuli. However,...
The homotetrameric thermosensitive transient receptor potential vanilloid 1-4 (TRPV1-4) channels in sensory neurons are strongly responsive to heat stimuli. However, their cold activations have not been reported in line with the nonzero heat capacity difference during heat or cold unfolding transitions. Here, along with the experimental examinations of the predicted ring size changes in different domains against the central pore during channel gating at various temperatures, the K169A mutant of reduced human TRPV3 was first found to be activated and inactivated by cold below 42°C. Further thermoring analyses revealed distinct heat and cold unfolding pathways, which resulted in different protein thermostabilities. Thus, both cold and heat unfolding transitions of thermosensitive TRPV1-4 channels may exist once a mutation destabilizes the closed state.
PubMed: 38746116
DOI: 10.21203/rs.3.rs-4285061/v1 -
DNA Repair Jul 2024The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including... (Review)
Review
The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including the removal of DNA-protein crosslinks (DPCs) from the DNA. Growing evidence demonstrates in the last years that Cdc48/p97 is pivotal in targeting ubiquitinated and SUMOylated substrates on chromatin, thereby supporting the DNA damage response. Along with its cofactors, notably Ufd1-Npl4, Cdc48/p97 has emerged as a central player in the unfolding and processing of DPCs. This review introduces the detailed structure, mechanism and cellular functions of Cdc48/p97 with an emphasis on the current knowledge of DNA-protein crosslink repair pathways across several organisms. The review concludes by discussing the potential therapeutic relevance of targeting p97 in DPC repair.
Topics: Valosin Containing Protein; DNA Repair; Humans; Saccharomyces cerevisiae Proteins; DNA; Saccharomyces cerevisiae; DNA Damage; Cell Cycle Proteins; Nuclear Proteins; Adenosine Triphosphatases; DNA-Binding Proteins; Animals; Intracellular Signaling Peptides and Proteins
PubMed: 38744091
DOI: 10.1016/j.dnarep.2024.103691 -
The Journal of Physical Chemistry. B May 2024In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex...
In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex biomacromolecules. We have previously reported on the folding/unfolding kinetics of a model α-helix. Here, we study folding transitions in chignolin (GYDPETGTWG), a short β-hairpin peptide previously used as a model to study conformational changes in β-sheet proteins. Although previously suggested, until now, the role of the Tyr2-Trp9 interaction in the folding mechanism of chignolin was not clear. In the present work, pH-dependent conformational changes of chignolin were characterized by circular dichroism (CD), nuclear magnetic resonance (NMR), ultrafast pH-jump coupled with time-resolved photoacoustic calorimetry (TR-PAC), and molecular dynamics (MD) simulations. Taken together, our results present a comprehensive view of chignolin's folding kinetics upon local pH changes and the role of the Tyr2-Trp9 interaction in the folding process. CD data show that chignolin's β-hairpin formation displays a pH-dependent skew bell-shaped curve, with a maximum close to pH 6, and a large decrease in β-sheet content at alkaline pH. The β-hairpin structure is mainly stabilized by aromatic interactions between Tyr2 and Trp9 and CH-π interactions between Tyr2 and Pro4. Unfolding of chignolin at high pH demonstrates that protonation of Tyr2 is essential for the stability of the β-hairpin. Refolding studies were triggered by laser-induced pH-jumps and detected by TR-PAC. The refolding of chignolin from high pH, mainly due to the protonation of Tyr2, is characterized by a volume expansion (10.4 mL mol), independent of peptide concentration, in the microsecond time range (lifetime of 1.15 μs). At high pH, the presence of the deprotonated hydroxyl (tyrosinate) hinders the formation of the aromatic interaction between Tyr2 and Trp9 resulting in a more disorganized and dynamic tridimensional structure of the peptide. This was also confirmed by comparing MD simulations of chignolin under conditions mimicking neutral and high pH.
Topics: Hydrogen-Ion Concentration; Kinetics; Protein Folding; Molecular Dynamics Simulation; Oligopeptides; Protein Structure, Secondary
PubMed: 38733339
DOI: 10.1021/acs.jpcb.3c08271 -
Foods (Basel, Switzerland) Apr 2024Spirulina protein (SP) is recognized as a nutritious edible microbial protein and holds potential as a natural emulsifier. Due to the inherent challenges SP faces in...
Spirulina protein (SP) is recognized as a nutritious edible microbial protein and holds potential as a natural emulsifier. Due to the inherent challenges SP faces in stabilizing high internal phase emulsions (HIPEs), ultrasonic techniques were utilized for modification. Noticeable alterations in the structural and functional properties of SP were observed following ultrasonic treatment at various power levels (0, 100, 300, and 500 W). Ultrasound treatment disrupted non-covalent interactions within the protein polymer structure, leading to the unfolding of molecular structures and the exposure of hydrophobic groups. Importantly, the particle size of SP was reduced the most at an ultrasonic power of 300 W, and the three-phase contact angle reached its peak at 84.3°. The HIPEs stabilized by SP modified with 300 W ultrasonication have high apparent viscosity and modulus values and strong storage stability under different environmental conditions. Additionally, the encapsulation of curcumin in HIPEs led to improved retention of curcumin across various settings. The bioavailability increased to 35.36, which is 2.8 times higher than the pure oil. These findings suggest that ultrasound-modified SP is a promising emulsifier for HIPEs, and is expected to encapsulate hydrophobic nutrients such as curcumin more effectively.
PubMed: 38731694
DOI: 10.3390/foods13091324 -
Food Science & Nutrition May 2024In contrast to its widespread traditional and popular culinary use to reduce weight, (adzuki beans) was not subjected to sufficient scientific scrutiny. Particularly,...
In contrast to its widespread traditional and popular culinary use to reduce weight, (adzuki beans) was not subjected to sufficient scientific scrutiny. Particularly, its saponins whose role was never investigated before to unveil the beans' antidiabetic and anti-obesity effects. Four vital pancreatic and intestinal carbohydrate enzymes were selected to assess the potency of the triterpenoidal saponins of to bind and activate these proteins through high-precision molecular modeling and dynamics mechanisms with accurate molecular mechanics Generalized Born Surface Area (MMGBSA) energy calculations; thus, recognizing their anti-obesity potential. Our results showed that adzukisaponin VI and adzukisaponin IV were the best compounds in the -amylase and -glucosidase enzymatic grooves, respectively. Adzukisaponin VI and angulasaponin C were the best fitting in the N-termini of sucrase-isomaltose (SI) enzyme, and angulasaponin C was the best scoring compound in maltase-glucoamylase C-termini. All of them outperformed the standard drug acarbose. These compounds in their protein complexes were selected to undergo molecular simulations of the drug-bound protein compared to the apo-protein through 100 ns, which confirmed the consistency of binding to the key amino acid residues in the four enzyme pockets with the least propensity of unfolding. Detailed analysis is given of the different polar and hydrophobic binding interactions of docked compounds. While maltase-adzukisaponin VI complex scored the lowest MMGBSA free energy of -67.77 Kcal/mol, α-amylase complex with angulasaponin B revealed the free binding energy of -74.18 Kcal/mol with a dominance of van der Waals energy (ΔEVDW) and the least change from the start to the end of the simulation time. This study will direct researchers to the significance of isolating the pure adzuki saponin components to conduct future in vitro and in vivo experimental works and even clinical trials.
PubMed: 38726452
DOI: 10.1002/fsn3.4032 -
Protein Science : a Publication of the... Jun 2024One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining...
One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining the binding specificities of anti-amyloid β (Aβ) antibodies have limited ability to differentiate between complexes that form between antibodies and monomeric or oligomeric Aβ species during the dynamic Aβ aggregation process. Here, we present a high-resolution native ion-mobility mass spectrometry (nIM-MS) method to investigate complexes formed between a variety of Aβ oligomers and three Aβ-specific IgGs, namely two antibodies with relatively high conformational specificity (aducanumab and A34) and one antibody with low conformational specificity (crenezumab). We found that crenezumab primarily binds Aβ monomers, while aducanumab preferentially binds Aβ monomers and dimers and A34 preferentially binds Aβ dimers, trimers, and tetrameters. Through collision induced unfolding (CIU) analysis, our data indicate that antibody stability is increased upon Aβ binding and, surprisingly, this stabilization involves the Fc region. Together, we conclude that nIM-MS and CIU enable the identification of Aβ antibody binding stoichiometries and provide important details regarding antibody binding mechanisms.
Topics: Amyloid beta-Peptides; Antibodies, Monoclonal, Humanized; Ion Mobility Spectrometry; Humans; Mass Spectrometry; Protein Binding; Antibodies, Monoclonal; Protein Multimerization
PubMed: 38723181
DOI: 10.1002/pro.5008