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Protein and Peptide Letters Mar 2014In this work, we review the process of protein unfolding characterized by a solid-state nanopore based device. The occupied or excluded volume of a protein molecule in a... (Review)
Review
In this work, we review the process of protein unfolding characterized by a solid-state nanopore based device. The occupied or excluded volume of a protein molecule in a nanopore depends on the protein's conformation or shape. A folded protein has a larger excluded volume in a nanopore thus it blocks more ionic current flow than its unfolded form and produces a greater current blockage amplitude. The time duration a protein stays in a pore also depends on the protein's folding state. We use Bovine Serum Albumin (BSA) as a model protein to discuss this current blockage amplitude and the time duration associated with the protein unfolding process. BSA molecules were measured in folded, partially unfolded, and completely unfolded conformations in solid-state nanopores. We discuss experimental results, data analysis, and theoretical considerations of BSA protein unfolding measured with silicon nitride nanopores. We show this nanopore method is capable of characterizing a protein's unfolding process at single molecule level. Problems and future studies in characterization of protein unfolding using a solid-state nanopore device will also be discussed.
Topics: Animals; Cattle; Models, Molecular; Nanopores; Protein Transport; Protein Unfolding; Serum Albumin, Bovine
PubMed: 24370259
DOI: 10.2174/09298665113209990077 -
Journal of Physics. Condensed Matter :... Dec 2012The conformation of protein molecules is determined by a balance of various forces, including van der Waals attraction, electrostatic interaction, hydrogen bonding, and... (Review)
Review
The conformation of protein molecules is determined by a balance of various forces, including van der Waals attraction, electrostatic interaction, hydrogen bonding, and conformational entropy. When protein molecules encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein molecule strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that protein conformation changes during the adsorption process due to the protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. This review considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces.
Topics: Adsorption; Kinetics; Models, Molecular; Protein Conformation; Protein Unfolding; Proteins
PubMed: 23164927
DOI: 10.1088/0953-8984/24/50/503101 -
Nanotechnology Jan 2024Protein sequencing is crucial for understanding the complex mechanisms driving biological functions. However, proteins are usually folded in their native state and the...
Protein sequencing is crucial for understanding the complex mechanisms driving biological functions. However, proteins are usually folded in their native state and the mechanism of fast protein conformation transitions still remains unclear, which make protein sequencing challenging. Molecular dynamics simulations with accurate force field are now able to observe the entire folding/unfolding process, providing valuable insights into protein folding mechanisms. Given that proteins can be unfolded, nanopore technology shows great potential for protein sequencing. In this study, we proposed to use MoS/SnSheterostructures to firstly unfold proteins and then detect them by a nanopore in the heterostructural membrane. All-atom molecular dynamics simulations performed in this work provided rich atomic-level information for a comprehensive understanding of protein unfolding process and mechanism on the MoS/SnSheterostructure, it was found that the strong binding of protein to SnSnanostripe and hydrogen bond breaking were the main reasons for unfolding the protein on the heterostructure. After the protein was fully unfolded, it was restrained on the nanostripe because of the affinity of protein to the SnSnanostripe. Thus by integrating the proposed unfolding technique with nanopore technology, detection of linear unfolded peptide was realized in this work, allowing for the identification of protein components, which is essential for sequencing proteins in the near future.
Topics: Molybdenum; Nanopores; Protein Folding; Protein Unfolding; Proteins
PubMed: 38118165
DOI: 10.1088/1361-6528/ad177f -
Protein Science : a Publication of the... Jan 2021Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited...
Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature T , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations.
Topics: Crystallography, X-Ray; Hot Temperature; Membrane Proteins; Multiprotein Complexes; Protein Folding; Protein Unfolding; Software
PubMed: 33140490
DOI: 10.1002/pro.3986 -
Biophysical Journal Jun 2021Every amino acid residue can influence a protein's overall stability, making stability highly susceptible to change throughout evolution. We consider the distribution of...
Every amino acid residue can influence a protein's overall stability, making stability highly susceptible to change throughout evolution. We consider the distribution of protein stabilities evolutionarily permittable under two previously reported protein fitness functions: flux dynamics and misfolding avoidance. We develop an evolutionary dynamics theory and find that it agrees better with an extensive protein stability data set for dihydrofolate reductase orthologs under the misfolding avoidance fitness function rather than the flux dynamics fitness function. Further investigation with ribonuclease H data demonstrates that not any misfolded state is avoided; rather, it is only the unfolded state. At the end, we discuss how our work pertains to the universal protein abundance-evolutionary rate correlation seen across organisms' proteomes. We derive a closed-form expression relating protein abundance to evolutionary rate that captures Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens experimental trends without fitted parameters.
Topics: Evolution, Molecular; Humans; Protein Folding; Protein Stability; Protein Unfolding; Proteome; Saccharomyces cerevisiae
PubMed: 33932438
DOI: 10.1016/j.bpj.2021.03.042 -
Molecules (Basel, Switzerland) Feb 2022Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are... (Review)
Review
Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are related to its unfolding processes induced by physical or chemical treatments. Unfolding occurs through a number of individual steps, generating transient intermediates through reversible and irreversible modifications. The rate of formation of these intermediates and of their further evolution into different structures often dictates the outcome of a given process. This report addresses the main structural features of the BLG unfolding intermediates under conditions that may facilitate or impair their formation in response to chemical or physical denaturing agents. In consideration of the short lifespan of the transient species generated upon unfolding, this review also discusses how various methodological approaches may be adapted in exploring the process-dependent structural modifications of BLG from a kinetic and/or a thermodynamic standpoint. Some of the conceptual and methodological approaches presented and discussed in this review can provide hints for improving the understanding of transient conformers formation by proteins present in other food systems, as well as when other physical or chemical denaturing agents are acting on proteins much different from BLG in complex food systems.
Topics: Animals; Cattle; Lactoglobulins; Milk; Models, Molecular; Protein Denaturation; Protein Stability; Protein Unfolding; Thermodynamics
PubMed: 35164393
DOI: 10.3390/molecules27031131 -
The Journal of Physical Chemistry. B Feb 2022Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the...
Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.
Topics: Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Protein Denaturation; Protein Folding; Protein Unfolding; Proteins; Thermodynamics
PubMed: 35128921
DOI: 10.1021/acs.jpcb.1c10750 -
Nature Nanotechnology Nov 2023
Topics: Nanopores; Proteins; Amino Acid Sequence; Protein Unfolding; High-Throughput Nucleotide Sequencing
PubMed: 37626147
DOI: 10.1038/s41565-023-01480-6 -
Methods in Molecular Biology (Clifton,... 2012We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of... (Review)
Review
We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of magnitude about transport timescales through pores, forces involved in unzipping processes … we present our experiments on DNA unzipping or protein unfolding using a nanopore. We point out the difficulties that can be encountered during these experiments, such as the signal analysis problems, noise issues, or experimental limitations of such system.
Topics: Algorithms; Bacterial Proteins; Biological Transport; Electroosmosis; Hemolysin Proteins; Maltose-Binding Proteins; Membranes, Artificial; Nanopores; Nucleic Acid Denaturation; Protein Unfolding
PubMed: 22528258
DOI: 10.1007/978-1-61779-773-6_4 -
Analytical Chemistry Nov 2022Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase () is an important method for the study of...
Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase () is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between and behavior, we use unbiased molecular dynamics (MD) to create models of unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded models for observed charge states of NTL9. The unfolding behavior replicates the behavior in-solution and is in line with the observations; however, the theoretical collision cross section (CCS) of the models was lower compared to that of the data, which may reflect reduced sampling.
Topics: Protons; Protein Unfolding; Molecular Dynamics Simulation; Proteins; Ions; Protein Conformation
PubMed: 36350278
DOI: 10.1021/acs.analchem.2c03352