Unusual cold denaturation of a small protein domain

A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.     

Transmembrane and extramembrane contributions to membrane protein thermal stability: studies with the NaChBac sodium channel

The thermal stabilities of the extramembranous and transmembranous regions of the bacterial voltage-gated sodium channel NaChBac have been characterised using thermal-melt synchrotron radiation circular dichroism (SRCD) spectroscopy. A series of constructs, ranging from the full-length protein containing both the C-terminal cytoplasmic and the transmembranous domains, to proteins with decreasing amounts of the cytoplasmic domain, were examined in order to separately define the roles of these two types of domains in the stability and processes of unfolding of a membrane protein. The sensitivity of the SRCD measurements over a wide range of wavelengths and temperatures has meant that subtle but reproducible conformational changes could be detected with accuracy. The residues in the C-terminal extramembranous domain were highly susceptible to thermal denaturation, but for the most part the transmembrane residues were not thermally-labile and retained their helical character even at very elevated temperatures. The process of thermal unfolding involved an initial irreversible unfolding of the highly labile distal extramembranous C-terminal helical region, which was accompanied by a reversible unfolding of a small number of helical residues in the transmembrane domain. This was then followed by the irreversible unfolding of a limited number of additional transmembrane helical residues at greatly elevated temperatures. Hence this study has been able to determine the different contributions and roles of the transmembrane and extramembrane residues in the processes of thermal denaturation of this multipass integral membrane protein.     

Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin

Single-molecule atomic force microscopy and spectroscopy were applied to detect molecular interactions stabilizing the structure of halorhodopsin (HR), a light-driven chloride pump from Halobacterium salinarum. Because of the high structural and sequence similarities between HR and bacteriorhodopsin, we compared their unfolding pathways and polypeptide regions that established structurally stable segments against unfolding. Unfolding pathways and structural segments stabilizing the proteins both exhibited a remarkably high similarity. This suggests that different amino acid compositions can establish structurally indistinguishable energetic barriers. These stabilizing domains rather result from comprehensive interactions of all amino acids within a structural region than from specific interactions. However, one additional unfolding barrier located within a short segment of helix E was detected for HR. This barrier correlated with a Pi-bulk interaction, which locally disrupts helix E and divides a structural stabilizing segment.     

Differential scanning calorimetric,circular dichroism,and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.     

A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.     

The thermal unfolding and domain structure of Na+/K+-exchanging ATPase. A scanning calorimetry study.

The thermal unfolding and domain structure of Na+/K+-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry (HS-DSC). The excess heat capacity function of Na+/K+-ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Td = 327.5 K was identified as corresponding to the beta subunit, while two other domains belong to the alpha subunit. The thermal unfolding of the low-temperature domain leads to large changes in the amplitude of the short-circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2-mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (approximately 12 J.g-1), suggesting that thermal denaturation of Na+/K+-ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.     

Three steps in the thermal unfolding of F-actin: an experimental evidence

The development of differential scanning calorimetry has resulted in an increased interest in studies of the unfolding process in proteins with the aim of identifying domains and interactions with ligands or other proteins. Several of these studies were done with actin and showed that the thermal unfolding of F-actin occurs in at least three steps; this was interpreted as the denaturation of independent domains. In the present work, we have followed the thermal unfolding of F-actin using differential scanning calorimetry (DSC), CD spectroscopy, and probe fluorescence. We found that the three steps revealed through DSC are not the denaturation of independent domains. These three steps are a change in the environment of cys 374 at 49.5 degrees C; a modification at the nucleotide-binding site at 55 degrees C; and the unfolding of the peptide chain at 64 degrees C. Previous interpretations of the thermograms of F-actin were thus erroneous. Since DSC is now widely used to study proteins, our experimental approach and conclusions may also be relevant in denaturation studies of proteins in general.     

Conformational stability of human erythrocyte transglutaminase. Patterns of thermal unfolding at acid and alkaline pH.

Tissue-type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0-9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition-state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH, we noted that transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-terminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C-terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N-terminal and C-terminal regions interact with each other in determining the structure of the native protein.     

Thermodynamic stability of a kappaI immunoglobulin light chain: relevance to multiple myeloma

Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry. The protein was found to contain a cysteinyl post-translational modification at Cys(214). Protein stability and conformation of MM-kappaI as a function of temperature or denaturant conditions at pH 7.4 and 4.8 were investigated. At pH 4.8, calorimetry demonstrated that MM-kappaI undergoes an incomplete, cooperative, partially reversible thermal unfolding with increased unfolding temperature and calorimetric enthalpy as compared to pH 7.4. Secondary and tertiary structural analyses provided evidence to support the presence of unfolding intermediates. Chemical denaturation resulted in more extensive protein unfolding. The stability of MM-kappaI was reduced and protein unfolding was irreversible at pH 4.8, thus suggesting that different pathways are utilized in thermal and chemical unfolding.     

The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy.

The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).     

Thermal unfolding molecular dynamics simulation of Escherichia coli dihydrofolate reductase: thermal stability of protein domains and unfolding pathway

Temperature induced unfolding of Escherichia coli dihydrofolate reductase was carried out by using molecular dynamic simulations. The simulations show that the unfolding generally involves an initial end-to-end collapse of the adenine binding domain into partially extended loops, followed by a gradual breakdown of the remaining beta sheet core structure. The core, which consists of beta strands 5-7, was observed to be the most resistant to thermal unfolding. This region, which is made up of part of the N terminus domain and part of the large domain of the E. coli dihydrofolate reductase, may constitute the nucleation site for protein folding and may be important for the eventual formation of both domains. The unfolding of different domains at different stages of the unfolding process suggests that protein domains vary in stability and that the rate at which they unfold can affect the overall outcome of the unfolding pathway. This observation is compared with the recently proposed hierarchical folding model. Finally, the results of the simulation were found to be consistent with a previous experimental study (Frieden, Proc Natl Acad Sci USA 1990;87:4413-4416) which showed that the folding process of E. coli dihydrofolate reductase involves sequential formation of the substrate binding sites.     

Thermal unfolding of small proteins with SH3 domain folding pattern

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12–16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. Proteins 31:309–319, 1998. © 1998 Wiley-Liss, Inc.     

Thermal effects in stretching of Go-like models of titin and secondary structures

The effect of temperature on mechanical unfolding of proteins is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. The behavior of the I27 domain of titin and its serial repeats is contrasted to that of simple secondary structures. In all cases, thermal fluctuations accelerate the unraveling process, decreasing the unfolding force nearly linearly at low temperatures. However, differences in bonding geometry lead to different sensitivity to temperature and different changes in the unfolding pattern. Due to its special native-state geometry, titin is much more thermally and elastically stable than the secondary structures. At low temperatures, serial repeats of titin show a parallel unfolding of all domains to an intermediate state, followed by serial unfolding of the domains. At high temperatures, all domains unfold simultaneously, and the unfolding distance decreases monotonically with the contact order, that is, the sequence distance between the amino acids that form the native contact.     

Different molecular mechanics displayed by titin's constitutively and differentially expressed tandem Ig segments

Titin is a giant elastic protein responsible for passive force generated by the stretched striated-muscle sarcomere. Passive force develops in titin's extensible region which consists of the PEVK segment in series with tandemly arranged immunoglobulin (Ig)-like domains. Here we studied the mechanics of tandem Ig segments from the differentially spliced (I65-70) and constitutive (I91-98) regions by using an atomic force microscope specialized for stretching single molecules. The mechanical stability of I65-70 domains was found to be different from that of I91-98 domains. In the range of stretch rates studied (0.05-1.00 microm/s) lower average domain unfolding forces for I65-70 were associated with a weaker stretch-rate dependence of the unfolding force, suggesting that the differences in the mechanical stabilities of the segments derive from differences in the zero force unfolding rate (K(0)(u)) and the characteristic distance (location of the barrier) along the unfolding reaction coordinate (DeltaX(u)). No effect of calcium was found on unfolding forces and persistence length of unfolded domains. To explore the structural basis of the differences in mechanical stabilities of the two fragment types, we compared the amino acid sequence of I65-70 domains with that of I91-98 domains and by using homology modeling analyzed how sequence variations may affect folding free energies. Simulations suggest that differences in domain stability are unlikely to be caused by variation in the number of hydrogen bonds between the force-bearing beta-strands at the domain's N- and C-termini. Rather, they may be due to differences in hydrophobic contacts and strand orientations.     

The multidomain structure of ceruloplasmin from calorimetric and limited proteolysis studies

Differential scanning calorimetry has been used to investigate the thermal stability of three different ceruloplasmins (from sheep, chicken, and turtle) in their native state and after limited proteolysis. The three undegraded proteins showed a similar structural organization in three calorimetric domains, although their temperature of unfolding varied from 57.8 degrees C (turtle) to 71.2 degrees C (sheep) to 82.1 degrees C (chicken). The spectroscopic and the catalytic properties were totally lost at temperatures corresponding to the unfolding of the less thermostable domain in the case of sheep and chicken ceruloplasmins and to the unfolding of the most thermostable domain in the turtle protein. Trypsin, but not plasmin, digestion caused a significant decrease of the thermal stability of sheep and chicken ceruloplasmins. Turtle ceruloplasmin was insensitive to both proteases. Comparing the thermodynamic parameters of the sheep protein in its undegraded and cleaved states revealed a mismatch between the three calorimetric domains and the 3-fold internal replication of the primary structure, which is evident in the highly homologous, fully sequenced human protein. Copper removal caused the rearrangement of the molecule in only two calorimetric domains, suggesting a role of the metal atoms in organizing a new calorimetric domain, which was tentatively assigned to the less thermostable cooperative unit of the native protein.     

Impact of deglycosylation and thermal stress on conformational stability of a full length murine igG2a monoclonal antibody: Observations from molecular dynamics simulations

With the rise of antibody based therapeutics as successful medicines, there is an emerging need to understand the fundamental antibody conformational dynamics and its implications towards stability of these medicines. Both deglycosylation and thermal stress have been shown to cause conformational destabilization and aggregation in monoclonal antibodies. Here, we study instabilities caused by deglycosylation and by elevated temperature (400 K) by performing molecular dynamic simulations on a full length murine IgG2a mAb whose crystal structure is available in the Protein Data bank. C^α‐atom root mean square deviation and backbone root mean square fluctuation calculations show that deglycosylation perturbs quaternary and tertiary structures in the C_H2 domains. In contrast, thermal stress pervades throughout the antibody structure and both Fabs and Fc regions are destabilized. The thermal stress applied in this study was not sufficient to cause large scale unfolding within the simulation time and most amino acid residues showed similar average solvent accessible surface area and secondary structural conformations in all trajectories. C_H3 domains were the most successful at resisting the conformational destabilization. The simulations helped identify aggregation prone regions, which may initiate cross‐β motif formation upon deglycosylation and upon applying thermal stress. Deglycosylation leads to increased backbone fluctuations and solvent exposure of a highly conserved APR located in the edge β‐strand A of the C_H2 domains. Aggregation upon thermal stress is most likely initiated by two APRs that overlap with the complementarity determining regions. This study has important implications for rational design of antibody based therapeutics that are resistant towards aggregation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Thermal unfolding simulations of bacterial flagellin: insight into its refolding before assembly

Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded β-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process.     

Gamma S-crystallin of bovine and human eye lens: solution structure, stability and folding of the intact two-domain protein and its separate domains

Human and bovine gammaS-crystallin (HgammaS and BgammaS) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E. coli. HgammaS and BgammaS are found to be authentic according to their spectral and hydrodynamic properties. Both full-length proteins and isolated domains are monomeric and exhibit high thermal and pH stabilities. The thermodynamic characterization made use of chemically and thermally-induced equilibrium unfolding transitions at varying pH. In spite of its exemplary two-domain structure, gammaS-crystallin does not show bimodal unfolding characteristics. In the case of BgammaS, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for HgammaS the opposite holds true. Differential scanning calorimetry confirms the results of chemically-induced equilibrium unfolding transitions. Over the whole pH range between 2.0 and 11.5, HgammaS-crystallin and its isolated domains (HgammaS-N and HgammaS-C) follow the two-state model. The two-state unfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains. Obviously, interactions between the domains do not contribute significantly to the overall stability of gammaS-crystallin. In contrast, the structurally closely related gammaB-crystallin owes much of its extreme stability to domain interactions.     

Quaternary structure of aldolase leads to differences in its folding and unfolding intermediates

Pulsed hydrogen exchange mass spectrometry has been used to investigate folding of rabbit muscle aldolase, an alpha/beta-barrel protein exhibiting the classic TIM structure. Aldolase unfolded in GdHCl refolded as the denaturant concentration was reduced by dialysis. Samples withdrawn during dialysis were pulse-labeled with deuterium to identify unfolded regions in structural forms highly populated during the folding process. Intact, labeled aldolase was digested into fragments, which were analyzed by HPLC electrospray ionization mass spectrometry to detect the H/D exchange along the aldolase backbone. For some concentrations of GdHCl, bimodal distributions of deuterium were found for most peptic fragments, indicating that regions represented by these fragments were either unfolded or folded in the intact polypeptide prior to labeling. The extent of folding was determined from these mass spectra, as well as by CD (220 nm) and enzymatic activity. These results show that folding to the active form involves three domains and two intermediates. Approximately 110 residues fold to highly compact forms in each step. These results also show that each folding domain includes widely separated regions of the backbone. When compared with the results of a previous study of aldolase unfolding, these results show that the folding and unfolding domains include most of the same residues. However, three short segments change domains depending on whether the process is folding or unfolding. These changes are attributed to the very stable quaternary structure of rabbit muscle aldolase.