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Analytical Chemistry May 2020Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass...
Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass spectrometry (MS) coupled with ion mobility spectrometry (IMS) is emerging as an important biophysical technique owing to its high sensitivity, rapid analysis time, and ability to interrogate sample complexity or heterogeneity and the ability to probe protein structure dynamics. Here, a commercial IMS-MS platform has been modified for static native ESI emitters and an extended mass-to-charge range (20 kDa /) and its performance capabilities and limits were explored for a range of protein and protein complexes. The results show new potential for this instrument platform for studies of large protein and protein complexes and provides a roadmap for extending the performance metrics for studies of even larger, more complex systems, namely, membrane protein complexes and their interactions with ligands.
Topics: Concanavalin A; Fructose-Bisphosphate Aldolase; Ion Mobility Spectrometry; Mass Spectrometry; Protein Conformation; Protein Unfolding; Streptavidin; Ubiquitin
PubMed: 32338885
DOI: 10.1021/acs.analchem.0c00772 -
Biochemistry Dec 2019Many proteins in cells and in the extracellular matrix assemble into force-bearing networks, and some proteins clearly transduce mechanical stimuli into biochemical... (Review)
Review
Many proteins in cells and in the extracellular matrix assemble into force-bearing networks, and some proteins clearly transduce mechanical stimuli into biochemical signals. Although structural mechanisms remain poorly understood, the designs of such proteins enable mechanical forces to either inhibit or facilitate interactions of protein domains with other proteins, including small molecules and enzymes, including proteases and kinases. Here, we review some of the structural proteins and processes that exhibit distinct modes of force-dependent signal conversion.
Topics: Animals; Biomechanical Phenomena; Humans; Protein Conformation; Protein Unfolding; Proteins
PubMed: 31736312
DOI: 10.1021/acs.biochem.9b00839 -
International Journal of Biological... Aug 2022Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of... (Review)
Review
Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of flexibility and rigidity are fundamental for understanding the relationships between function, structure and stability. Protein unfolding can often be triggered by thermal fluctuations with flexible residues usually on the protein surface. Therefore, identification and knowledge of the effect of modification to flexible regions in protein structures are required for efficient protein engineering and the rational design of thermally stable proteins. The most flexible regions in protein are loops, hence their rigidification is one of the effective strategies for increasing thermal stability. Directed evolution or rational design by computational prediction can also lead to the generation of thermally stable proteins. Computational protein design has been improved significantly in recent years and has successfully produced de novo stable backbone structures with optimized sequences and functions. This review discusses intramolecular and intermolecular interactions that determine the protein structure, and the strategies utilized in the mutagenesis of mesophilic proteins to stabilize and improve the functional characteristics of biocatalysts by describing efficient techniques and strategies to rigidify flexible loops at appropriate positions in the structure of the protein.
Topics: Protein Engineering; Protein Stability; Protein Unfolding; Proteins; Temperature
PubMed: 35772638
DOI: 10.1016/j.ijbiomac.2022.06.154 -
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 -
Biomacromolecules Nov 2023Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation...
Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.
Topics: Hydrogels; Protein Folding; Protein Stability; Kinetics; Temperature; Protein Denaturation
PubMed: 37906737
DOI: 10.1021/acs.biomac.3c00764 -
The Journal of Physical Chemistry. B Nov 2022The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an... (Review)
Review
The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an important field of research for the scientific community for the past five decades. Two important intermediate states, namely, wet molten globule (WMG) and dry molten globule (DMG), have emerged as critical milestones during protein folding-unfolding reactions. While much has been discussed about WMGs as a common unfolding intermediate, the evidence for DMGs has remained elusive owing to their near-native features, which makes them difficult to probe using global structural probes. This Review puts together the available literature and new evidence on DMGs to give a broader perspective on the universality of DMGs and discuss their significance in protein folding, function, and disease.
Topics: Protein Folding; Protein Unfolding; Protein Conformation; Circular Dichroism
PubMed: 36286394
DOI: 10.1021/acs.jpcb.2c04991 -
Biomolecules Feb 2020Most of the human diseases related to various proteopathies are confined to the brain, which leads to the development of various forms of neurological disorders. The...
Most of the human diseases related to various proteopathies are confined to the brain, which leads to the development of various forms of neurological disorders. The human brain consists of several osmolytic compounds, such as N-Acetylaspartate (NAA), myo-inositol (mI), glutamate (Glu), glutamine (Gln), creatine (Cr), and choline-containing compounds (Cho). Among these osmolytes, the level of NAA drastically decreases under neurological conditions, and, hence, NAA is considered to be one of the most widely accepted neuronal biomarkers in several human brain disorders. To date, no data are available regarding the effect of NAA on protein stability, and, therefore, the possible effect of NAA under proteopathic conditions has not been fully uncovered. To gain an insight into the effect of NAA on protein stability, thermal denaturation and structural measurements were carried out using two model proteins at different pH values. The results indicate that NAA increases the protein stability with an enhancement of structure formation. We also observed that the stabilizing ability of NAA decreases in a pH-dependent manner. Our study indicates that NAA is an efficient protein stabilizer at a physiological pH.
Topics: Aspartic Acid; Biomarkers; Brain Chemistry; Choline; Creatine; Glutamic Acid; Glutamine; Humans; Hydrogen-Ion Concentration; Inositol; Muramidase; Neurons; Osmosis; Protein Unfolding; Temperature; Thermodynamics
PubMed: 32059525
DOI: 10.3390/biom10020286 -
Journal of the American Chemical Society Feb 2022Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the electrical sensing realm of solid-state nanopores and is challenging due to...
Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the electrical sensing realm of solid-state nanopores and is challenging due to fast translocations and the use of high molar electrolytes. Despite providing an appreciable signal-to-noise ratio, high electrolyte concentrations can have adverse effects on the native protein structure. Herein, we present a thorough investigation of low electrolyte sensing conditions across a broad pH and voltage range generating conductive pulses (CPs) irrespective of protein net charge. We used Cas9 as the model protein and demonstrated that unfolding is noncooperative, represented by the gradual elongation or stretching of the protein, and sensitive to both the applied voltage and pH (i.e., charge state). The magnitude of unfolding and the isoelectric point (pI) of Cas9 was found to be correlated and a critical factor in our experiments. Electroosmotic flow (EOF) was always aligned with the transit direction, whereas electrophoretic force (EPF) was either reinforcing (pH < pI) or opposing (pH > pI) the protein's movement, which led to slower translocations at higher pH values. Further exploration of higher pH values led to slowing down of protein with > 30% of the population being slower than 0.5 ms. Our results would be critical for protein sensing at very low electrolytes and to retard their translocation speed without resorting to high-bandwidth equipment.
Topics: CRISPR-Associated Protein 9; Electroosmosis; Hydrogen-Ion Concentration; Isoelectric Point; Nanopores; Protein Conformation; Protein Unfolding
PubMed: 35143193
DOI: 10.1021/jacs.1c11540 -
Molecules (Basel, Switzerland) Oct 2022The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be... (Review)
Review
The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.
Topics: Protein Denaturation; Protein Conformation; Deuterium Oxide; Aryldialkylphosphatase; Electrophoresis, Polyacrylamide Gel; Cholinesterases; Biological Products; Thermodynamics; Protein Folding
PubMed: 36296453
DOI: 10.3390/molecules27206861 -
Science Advances May 2024Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially...
Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.
Topics: Protein Unfolding; Humans; Membrane Proteins; Aquaporin 1; Nuclear Magnetic Resonance, Biomolecular; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Folding; Kinetics; Thermodynamics
PubMed: 38758787
DOI: 10.1126/sciadv.adm7907