Tertiary Structural Transition Research Articles

In-droplet hydrogen-deuterium exchange to examine protein/peptide solution conformer heterogeneity.

Many different structure analysis techniques are not capable of probing the heterogeneity of solution conformations. Here, we examine the ability of in-droplet hydrogen-deuterium exchange (HDX) to directly probe solution conformer heterogeneity of a protein with mass spectrometry (MS) detection. Two vibrating capillary vibrating sharp-edge spray ionization (cVSSI) devices have been arranged such that they generate microdroplet plumes of the analyte and D2 O reagent, which coalesce to form reaction droplets where HDX takes place in the solution environment. The native HDX-MS setup has been first explored for two model peptides that have distinct structural compositions in solution. The effectiveness of the multidevice cVSSI-HDX in illustrating structural details has been further exploited to investigate coexisting solution-phase conformations of the protein ubiquitin. In-droplet HDX reveals decreased backbone exchange for a model peptide that has a greater helix-forming propensity. Differences in intrinsic rates of the alanine and serine residues may account for much of the observed protection. The data allow the first estimates of backbone exchange rates for peptides undergoing in-droplet HDX. That said, the approach may hold greater potential for investigating the tertiary structure and structural transitions of proteins. For ubiquitin protein, HDX reactivity differences suggest that multiple conformers are present in native solutions. The addition of methanol to buffered aqueous solutions of ubiquitin results in increased populations of solution conformers of higher reactivity. Data analysis suggests that partially folded conformers such as the A-state of ubiquitin increase with methanol content; the native state may be preserved to a limited degree even under stronger denaturation conditions. The deuterium uptake after in-droplet HDX has been observed to correspond to some degree with peptide backbone hydrogen protection based on differences in intrinsic rates of exchange. The presence of coexisting protein solution structures under native and denaturing solution conditions has been distinguished by the isotopic distributions of deuterated ubiquitin ions.

Interactions between the 5' UTR mRNA of the spe2 gene and spermidine regulate translation in S. pombe.

The 5′ untranslated regions (5′ UTR) of mRNAs play an important role in the eukaryotic translation initiation process. Additional levels of translational regulation may be mediated through interactions between structured mRNAs that can adopt interchangeable secondary or tertiary structures and the regulatory protein/RNA factors or components of the translational apparatus. Here we report a regulatory function of the 5′ UTR mRNA of the spe2 gene (SAM decarboxylase) in polyamine metabolism of the fission yeast Schizosaccharomyces pombe. Reporter assays, biochemical experiments, and mutational analysis demonstrate that this 5′ UTR mRNA of spe2 can bind to spermidine to regulate translation. A tertiary structure transition in the 5′ UTR RNA upon spermidine binding is essential for translation regulation. This study provides biochemical evidence for spermidine binding to regulate translation of the spe2 gene through interactions with the 5′ UTR mRNA. The identification of such a regulatory RNA that is directly associated with an essential eukaryotic metabolic process suggests that other ligand-binding RNAs may also contribute to eukaryotic gene regulation.

Interactions between SAM and the 5' UTR mRNA of the sam1 gene regulate translation in S. pombe.

The 5′ untranslated region (5′ UTR) of eukaryotic mRNA plays an important role in translation. Here we report the function of the 5′ UTR mRNA of S-adenosylmethionine synthetase (sam1) in translational modulation in the presence of SAM in fission yeast Schizosaccharomyces pombe. Reporter assays, binding and chemical probing experiments, and mutational analysis show that the 5′ UTR mRNA of sam1 binds to SAM to effect translation. Translational modulation is dependent on a tertiary structure transition in the RNA upon SAM binding. The characterization of such an RNA that is directly associated with an essential metabolic process in eukaryotes provides additional evidence that ligand binding by RNAs plays an important role in eukaryotic gene regulation.

Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris.

Streptokinase, a therapeutically important thrombolytic agent, is prone to C-terminal degradation and plasmin-mediated proteolytic processing. Since the protein was glycosylated during secretion from Pichia pastoris, therefore, the role of carbohydrate moieties on its stability was analyzed via in vivo blocking of N-glycosylation using tunicamycin where an increased degradation of streptokinase was observed. Further, the in vitro site-directed mutagenesis of the three putative N-glycosylation sites at asparagine residues 14, 265, and 377 to alanine revealed the essentiality of glycosylation of the 14th amino acid residue in its post-translational proteolytic stability without significantly affecting its biological activity. However, the mutation of both Asn265 and Asn377 did not seem to contribute toward its glycosylation but resulted in a 39% lower specific activity in case of the rSK-N265,377A. Moreover, the mutation of all three glycosylation positions drastically reduced the secretory expression of native streptokinase from 347 to 186.6mg/L for the triple mutant with a 14% lower specific activity of 56,738IU/mg from 65,808IU/mg. The secondary structure, tertiary structure, and thermal transition point (45-55°C) of all the deglycosylated variants did not show any significant differences when compared with fully glycosylated native streptokinase using CD and fluorescence spectroscopy. Furthermore, the longer acting plasmin-resistant variants were also developed via the mutation of lysine residues 59 and 386 to glutamine which enhanced its biological stability as a ~ 1.5-fold increase in the caseinolytic zone size was observed in case of rSK-K59Q and also in rSK-K59,386Q mutant without affecting the structural properties.

Domain swapping dissection in Thermotoga maritima arginine binding protein: How structural flexibility may compensate destabilization

Thermotoga maritima Arginine Binding Protein (TmArgBP) is a valuable candidate for arginine biosensing in diagnostics. This protein is endowed with unusual structural properties that include an extraordinary thermal/chemical stability, a domain swapped structure that undergoes large tertiary and quaternary structural transition, and the ability to form non-canonical oligomeric species. As the intrinsic stability of TmArgBP allows for extensive protein manipulations, we here dissected its structure in two parts: its main body deprived of the swapping fragment (TmArgBP20-233) and the C-terminal peptide corresponding to the helical swapping element. Both elements have been characterized independently or in combination using a repertoire of biophysical/structural techniques. Present investigations clearly indicate that TmArgBP20-233 represents a better scaffold for arginine sensing compared to the wild-type protein. Moreover, our data demonstrate that the ligand-free and the ligand-bound forms respond very differently to this helix deletion. This drastic perturbation has an important impact on the ligand-bound form of TmArgBP20-233 stability whereas it barely affects its ligand-free state. The crystallographic structures of these forms provide a rationale to this puzzling observation. Indeed, the arginine-bound state is very rigid and virtually unchanged upon protein truncation. On the other hand, the flexible ligand-free TmArgBP20-233 is able to adopt a novel state as a consequence of the helix deletion. Therefore, the flexibility of the ligand-free form endows this state with a remarkable robustness upon severe perturbations. In this scenario, TmArgBP dissection highlights an intriguing connection between destabilizing/stabilizing effects and the overall flexibility that could operate also in other proteins.

Inositol Hexakisphosphate Increases The Transition Constant And The Affinity Of Horse Aquomethemoglobin for 5,5′‐dithiobis(2‐nitrobenzoate)

CysF9[93]β exists in two tertiary conformations, r and t, which are in dynamic equilibrium. The equilibrium constant, Kequ, for the reaction of 5,5′‐dithiobis(2‐nitrobenzoate) — DTNB — with the CysF9[93]β sulfhydryl group of each hemoglobin was determined at 25°C as a function of pH. The reactivity of CysF9[93]β is affected by allosteric effectors such as the proton (H+) and inositol hexakisphosphate (inositol‐P6). The major and minor haemoglobins in horse haemolysate were separated using a column of Whatman's carboxymethylcellulose (CMC 52). The absorbance of the product of DTNB reaction, 5‐thio‐2‐nitrobenzoate, at different concentrations of DTNB and 50 μmol (haem) dm−3 was read at 412 nm. The absorbance was then substituted into an appropriate equation derived for the determination of the equilibrium constant, Kequ, for the reaction. Kequ decreased the orders of magnitude between pH 5.6 and 9.0 in the absence and presence of inositol‐P6. Inositol‐P6 increased the affinity of the major and minor Hb for DTNB but decreased the affinity of the minor aquomet‐Hb. The results show that inositol‐P6 increases the relative population of the t tertiary conformation. So, it increases the affinity of CysF9[93]β by changing the relative distribution of two protein conformations.Study objectiveThe study also seeks to find the equilibrium between the r and t isomers in the reaction of DTNB with CysF9[93]β sulphydryl group of the horse aquomethemoglobins. The relative populations of these two conformations from the pH dependence of equilibrium and kinetic parameters and the effect of inositol‐P6 on these relative populations were determined.MethodsPreparation and separation of horse hemoglobin had been described in Omotosho et al., 2017. Preparation and determination of aquomethemoglobin concentration: Aquomethemoglobin was prepared by the addition of a two‐fold excess of potassium ferricyanide, K3Fe(CN)6, to oxyhemoglobin of a known concentration. An approximately 1 mol dm−3 K3Fe(CN)6 solution was prepared by dissolving 0.33 g of the reagent in 1 cm3 of distilled water. The volume of the K3Fe(CN)6, v cm3, added to V cm3 of hemoglobin to give a two‐fold excess is related to the concentration of the hemoglobin solution.ResultsDomestic horse contains two hemoglobin types: A, the fast hemoglobin, and B, the slow hemoglobin. The two haemoglobins have identical α chains. Horse hemoglobin consists of two electrophoretically distinct components. From Figs. 1 and 2 it is seen that inositol‐P6 increases the affinity of CysF9[93]β of each haemoglobin for DTNB by about three orders of magnitude throughout the pH range 5.6 to 9.2.ConclusionFigs. 1 and 2 report the dependence of Kequ, the equilibrium constant of the reaction of DTNB with CysF9[93]β, on pH. The values of Kequ decrease by about two orders of magnitude for slow aquomet hemoglobin over the range 5.6 ≥ pH ≤ 9.0 whereas there in the fast hemoglobin, the values of Kequ increases by about six orders of magnitude and there is an increase in the affinity for the aquomet derivative. This shows that there is more insight on the nature of the tertiary structure transition in both T and R state hemoglobin.Support or Funding InformationWe appreciate Covenant University Centre for Research, Innovation and Development for the financial support.

The Unique Molecular Choreography of Giant Pore Formation by the Cholesterol-Dependent Cytolysins of Gram-Positive Bacteria.

The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant β-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophysical, and biochemical analyses has revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their β-barrel pore in eukaryotic membranes. Our knowledge of the molecular details of these dramatic structural changes in CDCs has transformed our understanding of how giant pore complexes are assembled and has been critical to our understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs are employed in pore-independent processes.

Tertiary Conformational Transition In Horse Haemoglobin Induced By Inositol Hexakisphosphate

The red blood cell of the domestic horse contains two haemoglobin types. The two haemoglobins were separated on a column of carboxymethylcellulose. The equilibrium constant, Kequ, for the reaction of 5,5′‐dithiobis(2‐nitrobenzoate) — DTNB — with the CysF9[93]β sulfhydryl group of each haemoglobin was determined at 25°C as a function of pH. The reactivity of CysF9[93]β is affected by allosteric effectors such as the proton (H+) and inositol hexakisphosphate (inositol‐P6). Between pH 5.6 and 9.0 Kequ decreased by about two to four orders of magnitude, demonstrating that H+ is a heterotropic allosteric effector of haemoglobin with respect to its reaction with DTNB. Inositol‐P6 also decreased Kequ by about two to four orders of magnitude across the experimental pH range. CysF9[93]β exists in two tertiary conformations, r and t, in dynamic equilibrium. Krt, the equilibrium constant for the r ↔ t conformational transition, was determined for each of the two horse haemoglobins from an analysis of the pH dependence of Kequ. The calculations from the pH dependence of Kequ showed that the pKa values of the ionisable groups coupled to the DTNB reaction vary between 5.0 and 8.9. The equilibrium constants, Krt, for the r↔t tertiary structure transition, were 0.143 ± 0.05 and 0.446 ± 0.22 for the fast and slow stripped horse haemoglobins respectively. In the presence of inositol‐P6, Krt for the fast and slow were 2.219 ± 0.79 and 2.214 ± 0.83 respectively. The results show that inositol‐P6 increases the relative population of the t tertiary conformation. So, it increases the affinity of CysF9[93]β by changing the relative distribution of two protein conformations.

Spectroscopic Studies on Unfolding Processes of Apo-Neuroglobin Induced by Guanidine Hydrochloride and Urea

Neuroglobin (Ngb), a recently discovered globin, is predominantly expressed in the brain, retina, and other nerve tissues of vertebrates. The unfolding processes of apo-neuroglobin (apoNgb) induced by guanidine hydrochloride (GdnHCl) and urea were investigated by spectroscopic methods. In the unfolding processes, apoNgb's tertiary structural transition was monitored by the changes of intrinsic fluorescence emission spectra, and its secondary structural transition was measured by the changes of far-ultraviolet circular dichroism (CD) spectra. In addition, 8-anilino-1-naphthalenesulfonic acid (ANS), a hydrophobic cluster binding dye, was also used to monitor the unfolding process of apoNgb and to explore its intermediates. Results showed that GdnHCl-induced unfolding of apoNgb was via a three-state pathway, that is, Native state (N) → Intermediate state (I) → Unfolded state (U), during which the intermediate was inferred by an increase in fluorescence intensity and the change of CD value. Gibbs free energy changes are 10.2 kJ·mol−1 for the first unfolding transition and 14.0 kJ·mol−1 for the second transition. However, urea-induced unfolding of apoNgb only underwent a two-state transition: Native state (N) → Partially unfolded state (P). The result showed that GdnHCl can efficiently affect the conformational states of apoNgb compared with those of urea. The work will benefit to have an understanding of the unfolding mechanism of apoNgb induced by GdnHCl and urea.

Comparison of High-Throughput Biophysical Methods to Identify Stabilizing Excipients for a Model IgG2 Monoclonal Antibody: Conformational Stability and Kinetic Aggregation Measurements

The overall conformational stability of a model IgG2 monoclonal antibody (mAb) was examined as a function of temperature and pH using an empirical phase diagram approach. Stabilizing excipients were then identified based on high-throughput methods including (1) kinetic studies measuring aggregation via increases in optical density and (2) thermally induced structural transitions as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The kinetic profiles of antibody aggregation at 65 °C were pH dependent and correlated well with pH effects on secondary and tertiary structural transitions due to heat stress. For the screening of stabilizing excipients, the inhibition of the rate of protein aggregation at pH 4.5 at 65°C, as represented by changes in optical density, was shown to have a clear trend with a modest correlation coefficient compared with the stabilizing effect of the same excipients on the conformational stability of the antibody as measured by DSC and tryptophan fluorescence spectroscopy. These results demonstrate the utility of combining high-throughput data from protein aggregation kinetic experiments and conformational stability studies to identify stabilizing excipients that minimize the physical degradation of an IgG2 mAb.

Ligand-Driven Vectorial Folding of Ribosome-Bound Human CFTR NBD1

The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood, aspect of protein conformational diseases. Here we use fluorescence resonance energy transfer (FRET) to characterize tertiary structural transitions of nascent polypeptides and show that the first nucleotide-binding domain (NBD1) of human CFTR, whose folding is defective in cystic fibrosis, folds via a cotranslational multistep pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389-500 emerge from the ribosome exit tunnel, initiating compaction of a small, N-terminal α/β-subdomain. Real-time kinetics of synchronized nascent chains revealed that subdomain folding is rapid, occurs coincident with synthesis, and is facilitated by direct ATP binding to the nascent polypeptide. These findings localize the major CF defect late in the NBD1 folding pathway and establish a paradigm wherein a cellular ligand promotes vectorial domain folding by facilitating an energetically favored local peptide conformation.

Allosteric Mechanism of Oxygen-Binding in Hemoglobin

The widely held structure-based mechanism of cooperative oxygen-binding in hemoglobin (Hb), in which the oxygen-affinity of Hb is regulated by the T to R-quaternary and associated tertiary structural transition, is based upon the assumption of the structure-function correlation: ([deoxy-state] =[T-quaternary structure]=[low-affinity state]) and ([oxy-state]=[R-quaternary structure]= [high-affinity state]. Simultaneous measurements of the quaternary structure by proton NMR and the oxygen-affinity by oxygen-binding equilibrium have yielded the following structural/functional states of Hb: (IV) T(deoxy)Hb with extreme low-affinity, (V) R(oxy)Hb with extreme low-affinity, and (VI) T(oxy)Hb with extreme low-affinity in the presence of potent heterotropic effectors as well as more conventional (II) T(deoxy)Hb with low-affinity and (III) R(oxy)Hb with high-affinity in the absence of heterotropic effectors. These results indicate the above-mentioned assumed structure-function correlation is no longer valid. The structure-based allosteric mechanism of Hb describes merely ligation-linked structural allostery rather than the cooperative mechanism of Hb. The structure and the function ( the oxygen-affinity, the cooperativty, and the Bohr effect) of Hb are regulated independently by a tug of war between the allosteric effects of oxygen (T-->R and increasing affinity) and the opposing allosteric effects of heterotropic effectors (T<--R and decreasing affinity) rather than the T to R-quaternary and associated tertiary structural transition. The oxygen-affinity is regulated by effector-linked dynamic structural changes.

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular β-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ∼2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.

Elucidation of structural changes in soy protein isolate upon heating by Raman spectroscopy

SummaryChanges in protein secondary and tertiary structure of soy protein isolate (SPI) upon heating process were investigated by Raman spectroscopy. SPI refrigerated and heated at 70, 100 and 200 °C was analysed. Non‐significant differences in secondary structure (α‐helix, β‐sheet, unordered and turn) were observed in SPI upon heating at these temperatures. However, comparison of the Raman spectra of SPI refrigerated and heated at 70, 100 and 200 °C reveals changes in hydrophobic group environments. An intensity decreasing trend of the 1340 cm−1 band attributable to tryptophan vibrations was observed, which involves an increase in solvent exposure of tryptophan residues. The spectral results also showed intensity increasing trends for the 1450 and 2935 cm−1 bands of SPI upon heating, which can be ascribed to breaking of hydrophobic contacts and subsequent solvent exposure of the corresponding hydrophobic aliphatic groups. Finally, it was found tertiary structural transitions towards greater proportions of buried tyrosine residues upon heating of SPI.

Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail ( Cortunix cortunix japonica)

We demonstrate that 5,5′-dithiobis(2-nitrobenzoate) – DTNB – reacts with only CysF9[93]β and CysB5[23]β among the multiple sulfhydryl groups of the major and minor hemoglobins of the Japanese quail ( Cortunix cortunix japonica). K equ, the equilibrium constant for the reaction, does not differ very significantly between the two hemoglobins. It decreases 430-fold between pH ≈ 5.6 and pH ≈ 9: from a mean of 7 ± 1 to a mean of 0.016 ± 0.003. Quantitative analyses of the K equ data based on published X-ray and temperature-jump evidence for a tertiary structure transition in liganded hemoglobin enable the calculation of K rt, the equilibrium constant for the r ←→ t tertiary structure transition. K rt differs significantly between the two hemoglobins: 0.744 ± 0.04 for the major, 0.401 ± 0.01 for the minor hemoglobin. The mean p K as of the two groups whose ionizations are coupled to the DTNB reaction are about the same as previously reported for mammalian hemoglobins.

Relation of Enzyme Activity to Local/Global Stability of Murine Adenosine Deaminase: 19F NMR Studies

Adenosine deaminase (ADA, EC 3.5.4.4) is a ubiquitous (beta/alpha)8-barrel enzyme crucial for purine metabolism and normal immune competence. In this study, it was observed that loss of enzyme activity of murine ADA (mADA) precedes the global secondary and tertiary structure transition when the protein is exposed to denaturant. The structural mechanism for this phenomenon was probed using site-specific 19F NMR spectroscopy in combination with [6-19F]tryptophan labeling and inhibitor binding. There are four tryptophan residues in mADA and all are located more than 12 A from the catalytic site. The 19F NMR spectra of [6-19F]Trp-labelled mADA show that the urea-induced chemical shift change of 19F resonance of W161, one of the four tryptophan 19F nuclei, correlates with the loss of enzyme activity. The urea-induced chemical shift change of another 19F resonance of W117 correlates with the change of the apparent rate constant for the binding of transition-state analogue inhibitor deoxycoformycin to the enzyme. On the other hand, the chemical environment of the local region around W264 does not change significantly, as a consequence of perturbation by low concentrations of urea or substrate analog. The results indicate that different regions of mADA have different local stability, which controls the activity and stability of the enzyme. The results provide new insights into the relationship between the function of a protein and its conformational flexibility as well as its global stability. This study illustrates the advantage of 19F NMR spectroscopy in probing site-related conformational change information in ligand binding, enzymatic activity and protein folding.

An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured.

Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far- and near-UV circular dichroism spectroscopy, one-dimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.

Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate.

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.

24] Application of circular dichroism to study RNA folding transitions

Circular dichroism (CD) spectroscopy has been widely used to study secondary structure formation in both proteins and nucleic acids. In this chapter, the application of CD is used to study RNA folding transitions. Modern commercially available CD spectrometers are capable of simultaneous CD and absorbance measurements. The amplitude of the applied photomultiplier voltage (high tension) directly relates to absorbance of the sample and is recorded along with the CD signal. This enables to measure transitions by both probes without any additional measurements. The chapter also discusses the use of CD in combination with Mg2+ or urea titrations to study the secondary and tertiary structural transitions in RNAs. Two-domain ribozyme P RNA have been from Bacillus subtilis folds with two distinct structural transitions, an unfolded to intermediate, U-to-I, transition and an intermediate to native transition. The U-to-I transition occurs at micromolar Mg2+ concentrations and is readily monitored by changes in absorbance (A260) and CD at 260 nm. These signals are primarily sensitive to the formation of helical structure. Tertiary transition occurs in millimolar Mg2+ range with a Hill coefficient of about 4. The formation of the tertiary structure of RNA is the result of structural changes involving the ribosephosphate backbone and non-Watson–Crick base pairing of nucleotides.

Complementary Analysis of Thermal Transition Multiplicity of Hen Egg-White Lysozyme at Low pH Using X-ray Scattering and Scanning Calorimetry

In this present paper we present a new method of small-angle X-ray scattering (SAXS) data analysis (called the TMA method) to examine multiplicity of structural transition of denaturation processes of proteins. The TMA method gives us a molar fraction of spatial-conformational-state and an indication of deviation from the two-state structural transition hypothesis in a structural transition process. Using this method we have successfully compared a spatial-conformational-state transition observed by SAXS with a thermodynamic-microstate transition observed by differential scanning calorimetry (DSC) in the thermal denaturation process of hen egg-white lysozyme (HEWL) at the pH range of 2.4−3.1. The TMA method is able to dissect spatial-conformational-state transition multiplicity depending on structural hierarchy. We have found that the thermal structural transition of HEWL is well-characterized mostly in terms of two different spatial-conformational-state transitions in the tertiary and intramolecular structures. Upon heating, the transition-midpoint temperature of the spatial-conformational-state transition of the tertiary structure mostly agrees with that of DSC, while the transition-midpoint temperature of the intramolecular structure greatly differs from that of DSC. The tertiary structural transition essentially obeys the two-state transition feature with a sharp critical temperature in comparison with the intramolecular structural transition. When the pH is lowered from 3.1 to 2.4, the former transition tends to shows a maximum deviation from the two-state transition feature around the transition temperature. On the other hand, the intramolecular structural transition proceeds continuously with a relatively large deviation in the whole temperature range measured at all pH values. The present results obtained by the TMA method essentially agree with our previous report using standard SAXS analyses in HEWL system at different pH range of 7.0−1.2, and would afford us further aspects to discuss experimental evidences with theoretical models of protein folding, such as nucleation-collapse models.