Cold Denaturation Research Articles

Instability Challenges and Stabilization Strategies of Pharmaceutical Proteins.

Maintaining the structure of protein and peptide drugs has become one of the most important goals of scientists in recent decades. Cold and thermal denaturation conditions, lyophilization and freeze drying, different pH conditions, concentrations, ionic strength, environmental agitation, the interaction between the surface of liquid and air as well as liquid and solid, and even the architectural structure of storage containers are among the factors that affect the stability of these therapeutic biomacromolecules. The use of genetic engineering, side-directed mutagenesis, fusion strategies, solvent engineering, the addition of various preservatives, surfactants, and additives are some of the solutions to overcome these problems. This article will discuss the types of stress that lead to instabilities of different proteins used in pharmaceutics including regulatory proteins, antibodies, and antibody-drug conjugates, and then all the methods for fighting these stresses will be reviewed. New and existing analytical methods that are used to detect the instabilities, mainly changes in their primary and higher order structures, are briefly summarized.

Mechanistic Insights into IscU Conformation Regulation for Fe-S Cluster Biogenesis Revealed by Variable Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry.

Iron-sulfur (Fe-S) cluster (ISC) cofactors are required for the function of many critical cellular processes. In the ISC Fe-S cluster biosynthetic pathway, IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. Interestingly, the form of IscU that binds IscS and functions in Fe-S cluster assembly remains controversial. Here, results from variable temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) establish that IscU exists in structured, intermediate, and disordered forms that rearrange to more extended conformations at higher temperatures. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding attenuates the cold/heat denaturation of IscU, promotes refolding of IscU, favors the structured and intermediate conformations, and inhibits the disordered high charge states. Overall, these findings provide a structural rationalization for the role of Zn(II) in stabilizing IscU conformations and IscS in altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nIM-MS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding.

Protein Unfolding in Freeze Frames: Intermediate States are Revealed by Variable-Temperature Ion Mobility-Mass Spectrometry.

The gas phase is an idealized laboratory for the study of protein structure, from which it is possible to examine stable and transient forms of mass-selected ions in the absence of bulk solvent. With ion mobility–mass spectrometry (IM-MS) apparatus built to operate at both cryogenic and elevated temperatures, we have examined conformational transitions that occur to the monomeric proteins: ubiquitin, lysozyme, and α-synuclein as a function of temperature and in source activation. We rationalize the experimental observations with a temperature-dependent framework model and comparison to known conformers. Data from ubiquitin show unfolding transitions that proceed through diverse and highly elongated intermediate states, which converge to more compact structures. These findings contrast with data obtained from lysozyme—a protein where (un)-folding plasticity is restricted by four disulfide linkages, although this is alleviated in its reduced form. For structured proteins, collision activation of the protein ions in-source enables subsequent “freezing” or thermal annealing of unfolding intermediates, whereas disordered proteins restructure substantially at 250 K even without activation, indicating that cold denaturation can occur without solvent. These data are presented in the context of a toy model framework that describes the relative occupancy of the available conformational space.

Temperature effects on hydrophobic interactions: Implications for protein unfolding

• Temperature effects on hydrophobic interactions are related to the solute size. • Temperature increase leads to the decrease of Rc (critical radius). • Protein stability may be related to temperature effects on hydrophobic interactions. • Molecular mechanism of cold denaturation may be different from heat unfolding. According to our recent studies on hydrophobic interactions, this work is devoted to investigate the temperature effects on hydrophobic interactions. Regarding to hydrophobic interactions, they are due to the structural competition between bulk and interfacial water. The water structure may be influenced by temperature, which undoubtedly affects the hydrophobic interactions. For the temperature effects on hydrophobic interactions, they are related to the size of solute. This is reflected on the dependence of Rc (critical radius) on temperature. With increasing temperature, it leads to the decrease of Rc. Additionally, this can be demonstrated by the calculated PMFs (potential mean force) between a pair of CH 4 molecules through MD (molecular dynamics) simulations. Based on the temperature effects on hydrophobic interactions, it is applied to understand the dependence of protein stability on temperature, such as cold denaturation and heat unfolding. Regarding to the molecular mechanism, cold denaturation is different from heat unfolding.

On the emerging of thawing drip: Role of myofibrillar protein renaturation

This study aimed to facilitate the understanding on the origin of thawing drip under different freezing rate. Eventually we observed significantly greater thaw loss produced by slow freezing (8.58%) as compared to fast freezing (6.41%) after 24 h of thawing. Back to the freezing, ice crystallization induced decline in pH and the cold denaturation of myofibrillar protein. However, independent of freezing rate, we noticed protein renaturation with pH restoring during thawing, evidenced by the decreasing surface hydrophobicity, increasing solubility and thermal stability, and gradually stabilized secondary structure. Meanwhile, the water-holding of myofibrils increased with thawing process along with the rising water mobility. Under fast freezing, the results indicated less extensive protein cold denaturation and lower water mobility during thawing. Besides, we proposed that the microenvironment of lower ionic strength in fast freezing should benefit the protein renaturation and water re-absorption, ultimately contributed to lower thaw loss.

Cold Denaturation of Proteins in the Absence of Solvent: Implications for Protein Storage.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Cold Denaturation of Proteins in the Absence of Solvent: Implications for Protein Storage.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable‐temperature ion mobility mass spectrometry (VT IM‐MS) allows us to measure the structure of molecules at sub‐ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep‐freezing minimizes denaturation and maintains the native fold and VT IM‐MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Recipes for Inducing Cold Denaturation in an Otherwise Stable Protein.

Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the freezing point of water. Understanding the mechanisms that determine cold denaturation and the rules that permit its observation is an important challenge. A way to approach them is to be able to induce cold denaturation in an otherwise stable protein by means of mutations. Here, we studied CyaY, a relatively stable bacterial protein with no detectable cold denaturation and a high melting temperature of 54 °C. We have characterized for years the yeast orthologue of CyaY, Yfh1, a protein that undergoes cold and heat denaturation at 5 and 35 °C, respectively. We demonstrate that, by transferring to CyaY the lessons learnt from Yfh1, we can induce cold denaturation by introducing a restricted number of carefully designed mutations aimed at destabilizing the overall fold and inducing electrostatic frustration. We used molecular dynamics simulations to rationalize our findings and demonstrate the individual effects observed experimentally with the various mutants. Our results constitute the first example of rationally designed cold denaturation and demonstrate the importance of electrostatic frustration on the mechanism of cold denaturation.

Heat and cold denaturation of yeast frataxin: The effect of pressure

Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This feature makes the protein particularly interesting for studies aiming at understanding the rules that determine protein fold stability. Here, we present the phase diagram of Yfh1 unfolding as a function of pressure (0.1–500 MPa) and temperature 278–313 K (5–40°C) both in the absence and in the presence of stabilizers using Trp fluorescence as a monitor. The protein showed a remarkable sensitivity to pressure: at 293 K, pressures around 10 MPa are sufficient to cause 50% of unfolding. Higher pressures were required for the unfolding of the protein in the presence of stabilizers. The phase diagram on the pressure-temperature plane together with a critical comparison between our results and those found in the literature allowed us to draw conclusions on the mechanism of the unfolding process under different environmental conditions.

A simple model of protein cold denaturation

Cold denaturation is a general feature of the conformational stability of globular proteins. Its modelization is still an important goal of biophysics. We present a modified version of the Schellman-Brandts model that is able to reproduce the occurrence of two conformational transitions, cold and hot denaturations. The model suggests that: (a) globular protein cooperativity can be obtained by distinguishing an inner core, buried from water contact, in a spherical globule; (b) the latter is stabilized by the hydrophobic effect that can be modelled by the Gibbs free energy gain associated with the burial of nonpolar side chains in the inner core; (c) cold denaturation comes from the decrease in the stabilization provided by the hydrophobic effect at low temperature.

Simple Cryoprotectant-Free Method to Advance Pulsed Dipolar ESR Spectroscopy for Capturing Protein Conformational Ensembles.

Double electron-electron resonance (DEER) is a powerful technique for studying protein conformations. To preserve the room-temperature ensemble, proteins are usually shock-frozen in liquid nitrogen prior to DEER measurements. The use of cryoprotectant additives is, therefore, necessary to ensure the formation of a vitrified state. Here, we present a simple modification of the freezing process using a flexible fused silica microcapillary, which increases the freezing rates and thus enables DEER measurement without the use of cryoprotectants. The Bid protein, which is highly sensitive to cryoprotectant additives, is used as a model. We show that DEER with the simple modification can successfully reveal the cold denaturation of Bid, which was not possible with the conventional DEER preparations. The DEER result reveals the nature of Bid folding. Our method advances DEER for capturing the chemically and thermally induced conformational changes of a protein in a cryoprotectant-free medium.

Molecular understanding of calorimetric protein unfolding experiments

Testing and predicting protein stability gained importance because proteins, including antibodies, became pharmacologically relevant in viral and cancer therapies. Isothermal scanning calorimetry is the principle method to study protein stability. Here, we use the excellent experimental heat capacity Cp(T) data from the literature for a critical inspection of protein unfolding as well as for the test of a new cooperative model. In the relevant literature, experimental temperature profiles of enthalpy, Hcal(T), entropy, Scal(T), and free energy, Gcal(T) are missing. First, we therefore calculate the experimental Hcal(T), Scal(T), and Gcal(T) from published Cp(T) thermograms. Considering only the unfolding transition proper, the heat capacity and all thermodynamic functions are zero in the region of the native protein. In particular, the free energy of the folded proteins is also zero and Gcal(T) displays a trapezoidal temperature profile when cold denaturation is included. Second, we simulate the DSC-measured thermodynamic properties with a new molecular model based on statistical-mechanical thermodynamics. The model quantifies the protein cooperativity and predicts the aggregate thermodynamic variables of the system with molecular parameters only. The new model provides a perfect simulation of all thermodynamic properties, including the observed trapezoidal Gcal(T) temperature profile. Importantly, the new cooperative model can be applied to a broad range of protein sizes, including antibodies. It predicts not only heat and cold denaturation but also provides estimates of the unfolding kinetics and allows a comparison with molecular dynamics calculations and quasielastic neutron scattering experiments.

Temperature Dependence of Hydrophobic and Hydrophilic Forces and Interactions.

Molecular dynamics simulations are used to compare the forces and Gibbs free energies associated with bringing small hydrophobic and hydrophilic solutes together in an aqueous solution at different temperatures between 280 and 360 °K. For the hydrophilic solutes, different relative orientations are used to distinguish between direct, intersolute hydrogen bonds (Hbond) and solutes simultaneously hydrogen bonding to a solvent water bridge. Interestingly, the temperature dependence of the hydrophobic and directly hydrogen bonding solutes turns out to be opposite to that of the bridged hydrophilic solutes, with the ΔG becoming more negative for the former and less negative for the latter with increasing temperature. Dissection of the free energy curves into enthalpy and entropy contributions, and further separation of the enthalpy term into solute-solute, solute-solvent, and solvent-solvent components provides insight into the physical molecular causes for the distinctive thermodynamic results. Finally, it is reasoned how the opposite temperature dependencies of the two types of hydrophilic interactions provide a rationale for the cold denaturation of proteins.

In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress.

Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice-liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as -15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near -10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (TCD ∼ -17 °C; calculated via isothermal chemical denaturation and Gibbs-Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near -5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

Applying text mining to identify relevant literature in food science: Cold denaturation as a case study.

In a research environment characterized by the five V's of big data, volume, velocity, variety, value, and veracity, the need to develop tools that quickly screen a large number of publications into relevant work is an increasing area of concern, and the data-rich food industry is no exception. Here, a combination of latent Dirichlet allocation and food keyword searches were employed to analyze and filter a dataset of 6102 publications about cold denaturation. After using the Python toolkit generated in this work, the approach yielded 22 topics that provide background and insight on the direction of research in this field, as well as identified the publications in this dataset which are most pertinent to the food industry with precision and recall of 0.419 and 0.949, respectively. Precision is related to the relevance of a paper in the filtered dataset and the recall represents papers which were not identified in the screening method. Lastly, gaps in the literature based on keyword trends are identified to improve the knowledge base of cold denaturation as it relates to the food industry. This approach is generalizable to any similarly organized dataset, and the code is available upon request. Practical Application: A common problem in research is that when you are an expert in one field, learning about another field is difficult, because you may lack the vocabulary and background needed to read cutting edge literature from a new discipline. The Python toolkit developed in this research can be applied by any researcher that is new to a field to identify what the key literature is, what topics they should familiarize themselves with, and what the current trends are in the field. Using this structure, researchers can greatly speed up how they identify new areas to research and find new projects.

Protein dynamics implications of the low- and high-temperature denaturation of myoglobin

We reinvestigate a simple model used in the literature concerning the thermodynamic analysis of protein cold denaturation. We derive an exact thermodynamic expression for cold denaturation and give a better approximation than exists in the literature for predicting cold denaturation temperatures in the two-state model. We discuss the "dark-side" implications of this work for previous temperature-dependent protein dynamics experiments and discuss microfluidic experimental technologies, which could explore the thermal stability range of proteins below the bulk freezing point of water.

The anatomy of unfolding of Yfh1 is revealed by site-specific fold stability analysis measured by 2D NMR spectroscopy.

Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

Light from a firefly at temperatures considerably higher and lower than normal

Bioluminescence emissions from a few species of fireflies have been studied at different temperatures. Variations in the flash-duration have been observed and interesting conclusions drawn in those studies. Here we investigate steady-state and pulsed emissions from male specimens of the Indian species Sclerotia substriata at temperatures considerably higher and lower than the ones at which they normally flash. When the temperature is raised to 34 °C, the peak wavelength gets red-shifted and the emitted pulses become the narrowest which broaden considerably thereafter for small increases in temperature; this probably indicates denaturation of the enzyme luciferase catalyzing the light-producing reaction. When the temperature is decreased to the region of 10.5–9 °C, the peak gets blue-shifted and the flash-duration increased abnormally with large fluctuation; this possibly implies cold denaturation of the luciferase. We conclude that the first or hot effect is very likely to be the reason of the species being dark-active on hot days, and the second or cold one is the probable reason for its disappearance at the onset of the winter. Our study makes the inference that these two happenings determine the temperature-tolerance, which plays a major role in the selection of the habitat for the firefly.

Cold denaturation induced helix-to-helix transition and its implication to activity of helical antifreeze protein

Study of cold denaturation is relevant for understanding the freeze tolerant activity of organism at sub-zero environment. The proteins responsible for the survival of these organisms contain abundance of alpha-helix. Here we study the effect of cold temperature on the alpha-helical region of a widely used model system, Trp-cage miniprotein by means of extensive atomistic molecular dynamics simulations in conjunction with replica exchange scheme. We find a helix-to-helix transition when the usual ~3.59 alpha-helix transforms to ~3.7 helix upon cooling below ~230 K. This transition is primarily driven by enthalpy gain from water-water interactions with the significant enhancement of low-density liquid (LDL)-like water at cold temperature. Because the transition from ~3.59-helix to ~3.7-helix occurs at cold temperature, it may be accompanied by cold denaturation of protein. Like the cold denatured state of Trp-cage, type-1 antifreeze proteins (AFP) also have ~3.7 or more residues per helical turn. However, unlike Trp-cage, AFP has the 3.7-helix in its biological active form. Such helical arrangement in AFP facilitates the hydrogen bonding with the surrounding clathrate or ice-like water molecules, which is crucial for the antifreeze activity of AFP.

Spatially resolved free-energy contributions of native fold and molten-globule-like Crambin

The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol−1 due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol−1. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.