Unfolding Midpoint Research Articles

Dark nanodiscs for evaluating membrane protein thermostability by differential scanning fluorimetry

Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the “dark” nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.

Stability Islands and the Folding Cooperativity of a Seven-Repeat Array from Topoisomerase V.

Cooperativity is a central feature of protein folding, but the thermodynamic and structural origins of cooperativity remain poorly understood. To quantify cooperativity, we measured guanidine-induced unfolding transitions of single helix-hairpin-helix (HhH)2 repeats and tandem pairs from a seven-repeat segment of Methanopyrus kandleri Topoisomerase V (Topo V) to determine intrinsic repeat stability and interfacial free energies between repeats. Most single-repeat constructs are folded and stable; moreover, several pairs have unfolding midpoints that exceed midpoints of the single repeats they comprise, demonstrating favorable coupling between repeats. Analyzing unfolding transitions with a modified Ising model, we find a broad range of intrinsic and interfacial free energies. Surprisingly, the G repeat, which lacks density in the crystal structure of Topo V without DNA, is the most stable repeat in the array. Using nuclear magnetic resonance spectroscopy, we demonstrate that the isolated G repeat adopts a canonical (HhH)2 fold and forms an ordered interface with the F-repeat but not with the H repeat. Using parameters from our paired Ising fit, we built a partition function for the seven-repeat array. The multistate unfolding transition predicted from this partition function is in excellent agreement with the experimental unfolding transition, providing strong justification for the nearest-neighbor model. The seven-repeat partition function predicts a native state in which three independent segments ("stability islands") of interacting repeats are separated by two unstable interfaces. We confirm this segmented architecture by measuring the unfolding transition of an equimolar mixture of these three separate polypeptides. This segmented structural organization may facilitate wrapping around DNA.

Planktonic adaptive evolution to the sea surface temperature in the Neoproterozoic inferred from ancestral NDK of marine cyanobacteria

The optimum growth temperature of ancestral cyanobacteria inhabiting the sea surface in the Neoproterozoic was estimated based on the thermal stability of experimentally reconstructed ancestral NDK enzymes. Ancestral NDKs of cyanobacteria that diversified ∼1.7, ∼1.0, ∼0.9, ∼0.7, ∼0.6, and ∼0.5 billion years ago were reconstructed and analyzed, and the unfolding midpoint temperatures (Tms) ranged from ∼65°C to ∼70°C. Among the host of analyzed NDKs, the ancestors of marine, planktonic α-cyanobacteria diversified ≤ ∼1.0 Ga are highly likely to have inhabited marine environments during the Neoproterozoic, while ancestral cyanobacteria diversified ∼1.7 billion years ago were possibly marine but the habitat is less constrained compared to the others. According to the calibration curves derived from extant organisms, the obtained Tms of α-cyanobacteria diversified ≤ ∼1.0 Ga correspond to the range of optimum growth temperatures of around ∼33–48°C. The temperature range agrees well with the long-term sea temperature trend during Neoproterozoic suggested by δ18O and δ30Si records from marine cherts. Adaptation to the low temperature during the snowball glaciations in the late Neoproterozoic was not observed, implying that adaptation of optimum growth conditions to the episodic low temperature may not have been necessary. Therefore, ancestral marine plankton must have consistently adapted to the interglacial sea surface temperature in the Neoproterozoic, which was approximately 5–20°C higher than that is today. They may have survived the glaciations by acquiring cold tolerance and/or by suppressing growth rate.

Reversible two-state folding of the ultrafast protein gpW under mechanical force

Ultrafast folding proteins have limited cooperativity and thus are excellent models to resolve, via single-molecule experiments, the fleeting molecular events that proteins undergo during folding. Here we report single-molecule atomic force microscopy experiments on gpW, a protein that, in bulk, folds in a few microseconds over a marginal folding barrier (∼1 kBT). Applying pulling forces of only 5 pN, we maintain gpW in quasi-equilibrium near its mechanical unfolding midpoint and detect how it interconverts stochastically between the folded and an extended state. The interconversion pattern is distinctly binary, indicating that, under an external force, gpW (un)folds over a significant free-energy barrier. Using molecular simulations and a theoretical model we rationalize how force induces such barrier in an otherwise downhill free-energy surface. Force-induced folding barriers are likely a general occurrence for ultrafast folding biomolecules studied with single-molecule force spectroscopy.

Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force.

The analysis and interpretation of single molecule force spectroscopy (smFS) experiments is often complicated by hidden effects from the measuring device. Here we investigate these effects in our recent smFS experiments on the ultrafast folding protein gpW, which has been previously shown to fold without crossing a free energy barrier in the absence of force (i.e., downhill folding). Using atomic force microscopy (AFM) smFS experiments, we found that a very small force of ∼5 pN brings gpW near its unfolding midpoint and results in two-state (un)folding patterns that indicate the emergence of a force-induced free energy barrier. The change in the folding regime is concomitant with a 30,000-fold slowdown of the folding and unfolding times, from a few microseconds that it takes gpW to (un)fold at the midpoint temperature to seconds in the AFM. These results are puzzling because the barrier induced by force in the folding free energy landscape of gpW is far too small to account for such a difference in time scales. Here we use recently developed theoretical methods to resolve the origin of the strikingly slow dynamics of gpW under mechanical force. We find that, while the AFM experiments correctly capture the equilibrium distance distribution, the measured dynamics are entirely controlled by the response of the cantilever and polyprotein linker, which is much slower than the protein conformational dynamics. This interpretation is likely applicable to the folding of other small biomolecules in smFS experiments, and becomes particularly important in the case of systems with fast folding dynamics and small free energy barriers, and for instruments with slow response times.

Complexity of the folding transition of the B domain of protein A revealed by the high-speed tracking of single-molecule fluorescence time series.

The equilibrium unfolding transition of the B domain of protein A (BdpA) was investigated by using single-molecule fluorescence spectroscopy based on line-confocal detection of fast-flowing samples. The method achieved the time resolution of 120 μs and the observation time of a few milliseconds in the single-molecule time-series measurements of fluorescence resonance energy transfer (FRET). Two samples of BdpA doubly labeled with donor and acceptor fluorophores, the first possessing fluorophores at residues 22 and 55 (sample 1) and the second at residues 5 and 55 (sample 2), were prepared. The equilibrium unfolding transition induced by guanidium chloride (GdmCl) was monitored by bulk measurements and demonstrated that the both samples obey the apparent two-state unfolding. In the absence of GdmCl, the single-molecule FRET measurements for the both samples showed a single peak assignable to the native state (N). The FRET efficiency for N shifts to lower values as the increase of GdmCl concentration, suggesting the swelling of the native state structure. At the higher concentration of GdmCl, the both samples convert to the unfolded state (U). Near the unfolding midpoint for sample 1, the kinetic exchange between N and U causes the averaging of the two states and the higher values of the relative fluctuation. The time series for different molecules in U showed slightly different FRET efficiencies, suggesting the apparent heterogeneity. Thus, the high-speed tracking of fluorescence signals from single molecules revealed a complexity and heterogeneity hidden in the apparent two-state behavior of protein folding.

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.

Human Phenotypically Distinct TGFBI Corneal Dystrophies Are Linked to the Stability of the Fourth FAS1 Domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

Thermodynamic basis of the thermostability of CYP175A1 from Thermus thermophilus

Detailed circular dichroism studies have been carried out to monitor thermal as well as denaturant induced unfolding of CYP175A1 from Thermus thermophilus and its mesophilic homologue, CYP101 from Pseudomonas putida. The unfolding midpoint temperatures for tertiary and secondary structures of the substrate-free CYP175A1 were found to be 83.7 degrees C and 87 degrees C respectively, while the corresponding midpoint temperatures for substrate-free CYP101 were at 47.7 degrees C and 51 degrees C respectively. The apparent C(m) value for GdnHCl induced unfolding of secondary structure of CYP175A1 was found to be 2.6M. The thermodynamic stability curves determined from GdnHCl induced unfolding of the enzymes at different temperatures, showed that CYP175A1 had higher free energy compared to CYP101 at temperature >10 degrees C. The results clearly established that the high thermostability of CYP175A1 arises predominantly due to higher enthalpy of the thermostable enzyme compared to CYP101. The larger number of salt bridges was proposed to be responsible for higher enthalpy of stabilization of CYP175A1.

Structural and Redox Behavior of OxyVita™, a Zero-linked Polymeric Hemoglobin: Comparison with Natural Acellular Polymeric Hemoglobins

A zero-linked polymeric hemoglobin (OxyVita Hb) has been developed for application as an acellular therapeutic hemoglobin-based-oxygen-carrier (HBOC). For effective and safe oxygen binding, transport and delivery, an HBOC must meet essential molecular requirements related to its structural integrity and redox stability. OxyVita is a super polymer possessing an average M.wt. of 17 x 10(6) Da. Structural integrity was determined by unfolding studies of OxyVita in the presence of increasing concentrations of urea. The unfolding midpoints (D(1/2)) of different preparations of OxyVita (solution and powder forms) were compared to Lumbricus Hb (LtHb) and Arenicola Hb (ArHb), natural acellular polymeric hemoglobins, which are serving as models for an effective and safe acellular HBOC. Reduction studies of OxyVita Hb using endogenous reducing agents were also investigated. Results from these studies indicate that: 1) OxyVita Hb exhibits greater resistance to conformational change than either LtHb or ArHb in the reduced (oxyHb) state; and 2) the reduction of met OxyVita Hb to oxyHb occurs slowly in the presence of either ascorbic acid (70% reduction in 560 min.) or beta-NADH (40% reduction in 90 min.). These studies provide consistent evidence that OxyVita Hb possesses physiochemical properties that exhibit structural integrity and redox behavior necessary for functioning as an effective and safe HBOC within clinical applications. These results are in agreement with observations made by other investigators as to the reduction in heme-loss of OxyVita Hb, essential for the reversible binding/release of molecular oxygen within the circulatory system.

Impact of cofactor on stability of bacterial (CopZ) and human (Atox1) copper chaperones

Here, we present the first characterization of in vitro unfolding and thermodynamic stability of two copper chaperone proteins: Bacillus subtilis CopZ and Homo sapiens Atox1 . We find that the unfolding reactions for apo- and Cu(I)-forms of CopZ and Atox1, induced by the chemical denaturant, guanidine hydrochloride (GuHCl), and by thermal perturbation are reversible two-state reactions. For both proteins, the unfolding midpoints shift to higher GuHCl concentrations and the thermodynamic stability is increased in the presence of Cu(I). Despite the same overall fold, apo-CopZ exhibits much lower thermal stability than apo-Atox1. Although the thermal stability of both proteins is increased in the presence of copper, the stabilizing effect is largest for the less stable variant. Divergent energetic properties of the apo- and holo-forms may be linked to conformational changes that facilitate copper transfer to the target.

Tuning λ 6–85 Towards Downhill Folding at its Melting Temperature

The five-helix bundle λ 6–85 * is a fast two-state folder. Several stabilized mutants have been reported to fold kinetically near-downhill or downhill. These mutants undergo a transition to two-state folding kinetics when heated. It has been suggested that this transition is caused by increased hydrophobicity at higher temperature. Here we investigate two histidine-containing mutants of λ 6–85 * to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable λHA is the fastest-folding lambda repressor to date ( k f − 1 ≈ k obs − 1 = 2.3 μs at 44 °C). It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. λHG has a weakened hydrophobic core. It is less stable than some slower folding mutants of λ 6–85 *, and it has more exposed hydrophobic surface area in the folded state. This mutant nonetheless folds very rapidly, and has the non-exponential folding kinetics of an incipient downhill folder even at the unfolding midpoint ( k m − 1 ≈ 2 μs, k a − 1 = 15 μs at 56 °C). We also compare the thermodynamic melting transition of λHG with the nominal two-state folding mutant λQG, which has a similar melting temperature. Unlike λQG, λHG yields fluorescence wavelength-dependent cooperativities and probe-dependent melting temperatures. This result combined with previous work shows that the energy landscapes of lambda repressor mutants support all standard folding mechanisms.

Probing site-specific conformational distributions in protein folding with solid-state NMR

We demonstrate an experimental approach to structural studies of unfolded and partially folded proteins in which conformational distributions are probed at a site-specific level by 2D solid-state 13C NMR spectroscopy of glassy frozen solutions. Experiments on chemical denaturation of the 35-residue villin headpiece subdomain, a model three-helix-bundle protein with a known folded structure, reveal that 13C-labeled residues in the three helical segments of the folded state have markedly different conformational distributions in the unfolded state. Moreover, the 2D solid-state NMR line shapes near the unfolding midpoint do not fit a simple two-state model, in which the conformational distributions of the unfolded component are assumed to be independent of denaturant concentration. Comparison with solid-state NMR spectra of peptides containing the individual helical segments suggests an alternative two-step description of conformational distributions in partially folded states of the helical villin headpiece subdomain, in which chemical denaturation is viewed as a disruption of tertiary contacts followed by equilibration of local secondary structure according to the intrinsic helical propensities of individual segments.

High‐throughput measurement of protein stability in microtiter plates

The direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition midpoint determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removal of proteins from the 96-well plate format in which they were expressed and purified. Herein we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability. Unfolding curves generated by the serial addition of denaturant into single wells allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C(1/2) +/- 0.05 M, m(G), and DeltaG(H2O)) for cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilized with various palmitate concentrations and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability.

High-pressure FTIR study of the stability of horseradish peroxidase. Effect of heme substitution, ligand binding, Ca++ removal, and reduction of the disulfide bonds.

The pressure stability of horseradish peroxidase isoenzyme C and the identification of possible stabilizing factors are presented. The effect of heme substitution, removal of Ca(2+), binding of a small substrate molecule (benzohydroxamic acid), and reduction of the disulfide bonds on the pressure stability were investigated by FTIR spectroscopy. HRP was found to be extremely stable under high pressure with an unfolding midpoint of 12.0 +/- 0.1 kbar. While substitution of the heme for metal-free mesoporphyrin did not change the unfolding pressure, Ca(2+) removal and substrate binding reduced the midpoint of the unfolding by 2.0 and 1.2 kbar, respectively. The apoprotein showed a transition as high as 10.4 kbar. However, the amount of folded structure present at the atmospheric pressure was considerably lower than that in all the other forms of HRP. Reduction of the disulfide bonds led to the least pressure stable form, with an unfolding midpoint at 9.5 kbar. This, however, is still well above the average pressure stability of proteins. The high-pressure stability and the analysis of the pressure-induced spectral changes indicate that the protein has a rigid core, which is responsible for the high stability, while there are regions with less stability and more conformational mobility.

Rapid gastric fluid digestion and biochemical characterization of engineered proteins enriched in essential amino acids.

The barley high lysine (BHL) proteins are nutritionally enhanced derivatives of barley chymotrypsin inhibitor-2 (CI-2). A compactly folded new CI-2 derivative, BHL9, was engineered with the highest content of threonine, tryptophan, and isoleucine yet achieved in this protein family (15.1, 9.4, and 12.1 wt %, respectively). BHL9 had an unfolding midpoint of 5.5 M guanidinium chloride, significantly greater than values for wild type (3.9 M) or for the previously most stable BHL protein, BHL8 (3.6 M). BHL9 and all other derivatives were digested within 15 s in simulated gastric fluid (SGF), suggesting nutritional availability upon ingestion. Denaturation of the proteins in SGF minus pepsin was revealed by changes in their fluorescence emission spectra and/or far UV circular dichroism spectra. The proteins lack homology to known allergens. Significantly, the BHL8 and BHL9 proteins were stable to proteases at pH 7.5 or 8.0, attesting to their potential for high expression in plants.

Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding.

Cytochrome c(552) from Hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of Escherichia coli. The b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 M lower than for the wild-type protein. The reduction potential is 75 mV lower than that of the recombinant wild-type protein. The heme can be removed from the b type variant, thus generating an apo protein that has, according to circular dichroism spectroscopy, an alpha-helical content different from that of the holo b type protein. The latter is readily reformed in vitro by addition of heme to the apo protein. This reforming suggests that previously observed assembly of cytochrome c(552), which has the typical class I cytochrome c fold, in the E. coli cytoplasm is a consequence of spontaneous thioether bond formation after binding of heme to a prefolded polypeptide. These observations have implications for the general problem of c type cytochrome biogenesis.

Unfolding and disassembly of the chaperonin GroEL occurs via a tetradecameric intermediate with a folded equatorial domain.

The chaperonin GroEL is a homotetradecamer in which the subunits (M(r) 57 000) are joined through noncovalent forces. This study reports on the unfolding and disassembly of GroEL in guanidine hydrochloride and urea. Kinetic and equilibrium measurements were made using amide hydrogen exchange/mass spectrometry, light scattering, and size-exclusion chromatography. Hydrogen exchange in GroEL destabilized in 1.8 M GdHCl (the unfolding midpoint is 1.2 M GdHCl) shows that the apical and intermediate domains unfold 3.1 times faster than the equatorial domain. Light scattering measurements made under the same conditions show that disassembly of the native GroEL tetradecamer occurs at the same rate as unfolding of the equatorial domain. This study of the kinetics of GroEL unfolding and disassembly demonstrates the existence of an intermediate that was identified as a tetradecamer with the apical and intermediate domains unfolded. Although this intermediate was easily detected in dynamic unfolding measurements, its population in equilibrium measurements at the midpoint for GroEL unfolding was too small to be detected. This study of GroEL unfolding and disassembly points to features that may be important in the folding and assembly of the GroEL macroassembly.

Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies.

Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.

Analysis of the conformation and stability of Escherichia coli derived-recombinant human interleukin 4 by circular dichroism

The conformation and stability of Escherichia coli derived recombinant human interleukin 4 (rhuIL-4) have been examined by circular dichroism (CD). Protein unfolding was detected by ellipticity changes at 222 nm with increasing concentrations of guanidine hydrochloride (GdnHCl). The unfolding midpoint ([GdnHCl]1/2) was 3.7 M, the free energy of unfolding, (delta GDH2O), was 5.9 kcal/mol and the dependence of delta GD on the GdnHCl concentration (m) was 1.6 (kcal/mol)/M. This unfolding was demonstrated to be reversible upon removal of the GdnHCl by dialysis. Analysis of the far-UV CD spectrum indicated the presence of a high percentage of alpha-helical structure (ca. 73%). A small change in ellipticity was noted over the pH range 1.9-9.6, suggesting that the protein undergoes a minor conformational change with an apparent pKa of 4.17. Virtually complete biological activity, measured in vitro in a T-cell proliferation assay, was recovered following exposure to extreme values of pH (i.e., pH 3 and 10). An analysis of the near-UV CD spectrum indicated that the single tryptophan residue at position 91 was unconstrained and most likely exposed to the solvent. Titration with 4,4'-dithiodipyridine and 2-nitrothiosulfobenzoate established that the six cysteine residues in rhuIL-4 were involved in intramolecular disulfide linkages. These data support that rhuIL-4 has a highly stable three-dimensional structure.