Equilibrium Unfolding Studies Research Articles

A Hydrophobic Core Stabilizes the Residual Structure in the RRM2 Intermediate State of the ALS-linked Protein TDP-43

Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.

A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.

Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3.

σB, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

The N-terminal MYB domains affect the stability and folding aspects of Arabidopsis thaliana MYB4 transcription factor under thermal stress.

The MYB4 transcription factor, a member of R2R3-type subfamily of MYB domain protein, plays a key role in the regulation of accumulation of UV-B absorbing phenylpropanoids in Arabidopsis. Although UV-B and thermal stress generate some common stress response, the effect of elevated temperature on the conformational stability of MYB4 remains limited. This study describes the folding and aggregation properties of Arabidopsis MYB4 protein under thermal stress condition. Circular dichroism spectral studies and Bis-ANS binding assays have indicated that the removal of the N-terminal MYB domain affects the structural conformation of the protein and disrupts surface hydrophobic binding sites at higher temperature. Urea-induced equilibrium unfolding studies revealed that the removal of the N-terminal region lowers the thermodynamic stability of MYB4 at elevated temperature. Tryptophan fluorescence spectral pattern and both in vitro and in vivo aggregation studies have revealed the importance of the N-terminal second MYB domain encompassing the N-terminal 62-116 amino acid residues in regulating MYB4 protein stability at higher temperature. On the other hand, comparison of the growth response of wild-type Arabidopsis and atmyb4 mutant line have suggested that MYB4 may not directly affect plant response under thermal stress condition and only marginal role of MYB4 in controlling thermomorphogenesis in Arabidopsis. Interestingly, immunoprecipitation studies have revealed that HSP90 specifically interacts with MYB4 in vivo at the endogenous level, indicating the possible role of HSP90 in governing the stability of MYB4 at elevated temperature in Arabidopsis.

Denaturant Induced Equilibrium Unfolding and Conformational Transitional Studies of Germinated Fenugreek β-Amylase Revealed Molten Globule like State at Low pH.

β-Amylase (EC 3.2.1.2) is a maltogenic enzyme, which releases β-maltose from the non-reducing end of the substrates. The enzyme plays important roles for the production of vaccine, maltiol and maltose rich syrups. Apart from these applications the enzyme protects cells from abiotic as well as oxidative damage. The enzyme is βwell characterized in βplants and microbes and crystal structures of β-amylases βhave been βobtained from sweet potato, soybean and Bacillus cereus. Find out correlation between structural and functional stability induced by change in pH, temperature and chaotropes. Activity, intrinsic fluorescence, extrinsic fluorescence, near- and far- ultraviolet circular dichroism spectroscopic measurements were performed. Peaks about 208 nm and 222 nm obtained by near-ultraviolet circular dichroism correspond to α-helix whereas peak at 215 nm shows presence of β-sheet. At pH 2.0, absence of tertiary structures, exposed of hydrophobic regions and presence of substantial secondary structures, revealed the existence of molten globule like state. Temperature induced denaturation studies showed that the enzyme was stable up to 75 ºC and the process was found to be irreversible in nature. Chaotropes dependent equilibrium unfolding studies revealed that at low concentration of chaotropes, ellipticity and intrinsic fluorescence βintensity were βdecreased βwhereas βenzymatic activity remained unchanged, which revealed fenugreek β-amylase is multi-domains enzyme and catalytic βdomain βis more βstable compare to non-catalytic domain. Moreover, the transition was sigmoidal and non-coincidental. Results indicate the probable existence of intermediate states that might perform significant role in physiological process and biotechnological applications.

The Effect of Chain Connectivity on the Thermodynamic, Kinetic and Mechanical Properties of Azurin

Azurin, a blue-copper protein, is responsible for electron transfer in Pseudomonas aeruginosa. It has widely been studied for its stability, electronic and electron transport properties, with potential applications in biomaterials-based devices. The amino acid chain connectivity is of fundamental importance in modulating the protein stability and folding. Hence, here we studied the thermodynamic, kinetic and mechanical properties of a circular permutant of apo-azurin (Azu-CPN42), with its N-terminus at Asn42, and compared them with those of the wild-type apo-azurin (WT-Azu). While the spectroscopic and copper-binding properties of Azu-CPN42 are unaltered, we observe changes in protein stability. Equilibrium unfolding studies using GdHCl suggest a drop of ∼0.6M in Cm and ∼13 kJ/mol in free energy of unfolding for Azu-CPN42 compared to that of WT-Azu. Kinetic studies reveal increase in the spontaneous unfolding rate by two-orders of magnitude, while the spontaneous folding rate being similar. Mechanical properties probed using single-molecule atomic force microscope suggest parallel unfolding pathways via 2- and 3-states, similar to that of WT-Azu reported previously. Interestingly, the intermediate stability is lower than that of the native state of Azu-CPN42 by ∼10pN, in accord with that of WT-Azu. As anticipated, the mechanical unfolding pathways of Azu-CPN42 and WT-Azu are different, as the two proteins are pulled along different N-C termini. This resulted in different mechanical intermediates, with a broad distribution of unfolding contour lengths (∼3-25 nm) for Azu-CPN42 while those of WT-Azu are centered at 7.5 ± 1.5 nm. This huge diversity may arise due to mechanical unraveling from both C- and N- termini as opposed to only C-terminal unraveling observed in WT-Azu. Our study reveals a decreased thermodynamic and kinetic stability and altered mechanical unfolding pathway of azurin upon changing its chain connectivity through circular permutation.

Human aldose reductase unfolds through an intermediate.

Background: Human aldose reductase (hAR) is the first and rate-limiting enzyme of the polyol pathway. For the development of secondary complications of diabetes in chronic hyperglycemic conditions, one of the critical factors is the increased flux of glucose through the polyol pathway. Due to this clinical implication, hAR attracted considerable attention from the drug discovery perspective. In spite of extensive characterization in the context of biochemical and structural aspects, we know very little about the unfolding behavior of hAR. This study reports equilibrium unfolding studies of hAR. Methods: We carried out thermal denaturation and chemical-induced equilibrium unfolding studies of hAR monitored by circular dichroism and fluorescence spectroscopy. Results: Thermal denaturation studies presented a classical picture of two-state unfolding from native to the denatured state. The data was used to derive thermodynamic parameters and study the thermostability of hAR. Chemical induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2.0 M of urea and GuHCl, respectively. Thermodynamic parameters derived from chemical-induced unfolding are in agreement with those obtained from thermal denaturation of hAR. Conclusion: This study revealed that aldose reductase unfolds from native to the unfolded state via an intermediate. Assessment of the thermodynamic stability of native, intermediate, and unfolded states shows that significant energy barriers separate these states, which ensures the cooperativity of unfolding. As hAR functions in cells that are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under the physiological state.

Human aldose reductase unfolds through an intermediate

Background: Human aldose reductase (hAR) is the first and rate-limiting enzyme of the polyol pathway. For the development of secondary complications of diabetes in chronic hyperglycemic conditions, one of the critical factors is the increased flux of glucose through the polyol pathway. Due to this clinical implication, hAR attracted considerable attention from the drug discovery perspective. In spite of extensive characterization in the context of biochemical and structural aspects, we know very little about the unfolding behavior of hAR. This study reports equilibrium unfolding studies of hAR. Methods: We carried out thermal denaturation and chemical-induced equilibrium unfolding studies of hAR monitored by circular dichroism and fluorescence spectroscopy. Results: Thermal denaturation studies presented a classical picture of two-state unfolding from native to the denatured state. The data was used to derive thermodynamic parameters and study the thermostability of hAR. Chemical induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2.0 M of urea and GuHCl, respectively. Thermodynamic parameters derived from chemical-induced unfolding are in agreement with those obtained from thermal denaturation of hAR. Conclusion: This study revealed that aldose reductase unfolds from native to the unfolded state via an intermediate. Assessment of the thermodynamic stability of native, intermediate, and unfolded states shows that significant energy barriers separate these states, which ensures the cooperativity of unfolding. As hAR functions in cells that are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under the physiological state.

An insight into the folding and stability of Arabidopsis thaliana SOG1 transcription factor under salinity stress in vitro

The present study describes the biophysical characterization of Arabidopsis thaliana SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) protein, a NAC domain transcription factor which plays central role in DNA damage response pathway, under salinity stress in vitro. Tryptophan fluorescence studies using purified recombinant wild type (full length) AtSOG1 and its N-terminal or C-terminal deletion forms (AtSOG1ΔNAC and AtSOG1ΔCT respectively) have revealed high salinity induced conformational change due to removal of the N-terminal NAC domain. Bis-ANS binding assays indicate that removal of the N-terminal NAC domain increases the surface hydrophobic binding sites, while the C-terminal region of SOG1 also plays important role in regulating the surface hydrophobicity aspects following exposure to high salinity. Circular dichroism (CD) spectral studies have indicated that removal of the N-terminal NAC domain affects the structural conformation of the protein under high salt concentration. Urea-induced equilibrium unfolding studies revealed decreased stability of C-terminal region due to removal of the N-terminal NAC domain. In vitro aggregation studies have indicated higher propensity of aggregation of AtSOG1ΔNAC due to salt treatment. Overall, our results provide evidence for the importance of both N-terminal NAC domain and the C-terminal region in regulating the stability of SOG1 protein under salinity stress in vitro.

Human aldose reductase unfolds through an intermediate.

Background: Human aldose reductase (hAR) converts glucose to sorbitol under hyperglycemic conditions. Aldose reductase is first and rate limiting enzyme of polyol pathway. Under hyperglycemia, increased flux of glucose through this pathway has been implicated in development of secondary complication in diabetes. Due to this clinical implication, aldose reductase attracted considerable attention from drug discovery perspective. In spite of extensive characterization of the biochemical and structural context, little is known about the unfolding behavior of aldose reductase. This study reports equilibrium unfolding studies of human aldose reductase. Methods: We carried out thermal and chemical induced equilibrium unfolding studies of human aldose reductase monitored by circular dichroism and tryptophan and ANS fluorescence spectroscopy. Results: Thermal unfolding studies present a classical picture of two state unfolding from native to unfolded state. The data was used to derive thermodynamic parameters and study thermostability of aldose reductase. Urea and GuHCl induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2 M of urea and GuHCl, respectively. Thermodynamic parameters from chemical induced unfolding are in agreement with those obtained from thermal unfolding. Conclusion: This study revealed that aldose reductase unfolds from native to unfolded state via an intermediate. Assessment of thermodynamic stability of native, intermediate and unfolded state shows that three states are separated by significant energy barriers that ensure cooperativity of unfolding. As hAR functions in cells which are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under physiological state.

Deciphering the Evolution of Cephalosporin Resistance to Ceftolozane-Tazobactam in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a class C β-lactamase (e.g., PDC-3) that robustly hydrolyzes early generation cephalosporins often at the diffusion limit; therefore, bacteria possessing these β-lactamases are resistant to many β-lactam antibiotics. In response to this significant clinical threat, ceftolozane, a 3' aminopyrazolium cephalosporin, was developed. Combined with tazobactam, ceftolozane promised to be effective against multidrug-resistant P. aeruginosa Alarmingly, Ω-loop variants of the PDC β-lactamase (V213A, G216R, E221K, E221G, and Y223H) were identified in ceftolozane/tazobactam-resistant P. aeruginosa clinical isolates. Herein, we demonstrate that the Escherichia coli strain expressing the E221K variant of PDC-3 had the highest minimum inhibitory concentrations (MICs) against a panel of β-lactam antibiotics, including ceftolozane and ceftazidime, a cephalosporin that differs in structure largely in the R2 side chain. The kcat values of the E221K variant for both substrates were equivalent, whereas the Km for ceftolozane (341 ± 64 µM) was higher than that for ceftazidime (174 ± 20 µM). Timed mass spectrometry, thermal stability, and equilibrium unfolding studies revealed key mechanistic insights. Enhanced sampling molecular dynamics simulations identified conformational changes in the E221K variant Ω-loop, where a hidden pocket adjacent to the catalytic site opens and stabilizes ceftolozane for efficient hydrolysis. Encouragingly, the diazabicyclooctane β-lactamase inhibitor avibactam restored susceptibility to ceftolozane and ceftazidime in cells producing the E221K variant. In addition, a boronic acid transition state inhibitor, LP-06, lowered the ceftolozane and ceftazidime MICs by 8-fold for the E221K-expressing strain. Understanding these structural changes in evolutionarily selected variants is critical toward designing effective β-lactam/β-lactamase inhibitor therapies for P. aeruginosa infections.IMPORTANCE The presence of β-lactamases (e.g., PDC-3) that have naturally evolved and acquired the ability to break down β-lactam antibiotics (e.g., ceftazidime and ceftolozane) leads to highly resistant and potentially lethal Pseudomonas aeruginosa infections. We show that wild-type PDC-3 β-lactamase forms an acyl enzyme complex with ceftazidime, but it cannot accommodate the structurally similar ceftolozane that has a longer R2 side chain with increased basicity. A single amino acid substitution from a glutamate to a lysine at position 221 in PDC-3 (E221K) causes the tyrosine residue at 223 to adopt a new position poised for efficient hydrolysis of both cephalosporins. The importance of the mechanism of action of the E221K variant, in particular, is underscored by its evolutionary recurrences in multiple bacterial species. Understanding the biochemical and molecular basis for resistance is key to designing effective therapies and developing new β-lactam/β-lactamase inhibitor combinations.

Inter and intra-subunit interactions at the subunit interface of chaperonin GroEL are essential for its stability and assembly

Chaperonin GroEL helps in the folding of substrate proteins under normal and stress conditions. Although it remains stable and functional during stress conditions, the quantitative estimation of stability parameters and the specific amino-acid residues playing a role in its stability are not known in sufficient detail. The reason for poor understanding is its large size, multimeric nature, and irreversible unfolding process. The X-ray crystal structure reveals that equatorial domain forms almost all intra and inter-subunit interactions for assembly of GroEL. Considering all these facts, we adopted alternate strategies to use monomeric GroEL, native GroEL and equatorial domain mutants (GroELK4E/GroELD523K/GroELD473C) to study the assembly and stability of GroEL. Loss of inter-subunit interaction involving K4 residue of one subunit and E59, I60, E61, I62 residues of adjacent subunit due to K4E mutation affect the oligomerization efficiency of GroEL subunits while the equilibrium unfolding studies on wild-type monomeric GroEL, native GroEL, and the selected mutants together demonstrate that intra-subunit interactions involving K4 and D523 of the same subunit play a critical role in the thermodynamic stability of both native and monomeric GroEL without affecting the oligomerization of subunits. The stability order between the GroELwild-type(M) and its variants is GroELwild-type(M) ≥ GroELD473C(M)˃GroELD523K(M)˃GroELK4E.

Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala19 can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor.

Investigation of folding unfolding process of a new variant of dihydrofolate reductase protein from Zebrafish

The folding and unfolding mechanisms of a small monomeric protein, Dihydrofolate reductase (1.5.1.3.) from a new variant, Zebrafish (zDHFR) has been studied through GdnHCl denaturation, followed by its refolding through dilution of the denaturant. Intrinsic and extrinsic fluorescence, far-UV CD and enzyme activity were employed to monitor structural and functional changes due to chemical denaturation. The unfolding transitions monitored by intrinsic fluorescence showed that GdnHCl based denaturation of zDHFR is reversible. At low concentration of the denaturant, zDHFR forms intermediate species as reflected by increased fluorescence intensity compared to the native and fully unfolded form. Equilibrium unfolding transition study of zDHFR induced by GdnHCl exhibited three- state process. The non- coincidence of fluorescence and far-UVCD based transitions curves support the establishment of three state model of zDHFR protein which involves native, intermediate and unfolded forms. Analysis of the equilibrium unfolding transition suggests the presence of non- native intermediate species. A comparative study of various species of DHFR shows that zDHFR has comparable thermodynamic stability with human counterpart and thus proved to be a good in vitro model system for structure- function relationship studies. Understanding various conformational states during the folding unfolding process of the zDHFR protein may provide important clues towards designing inhibitors against this important protein involved in cell cycle regulation.

Understanding the Physical and Molecular Basis of Stability of Arabidopsis DNA Pol λ under UV-B and High NaCl Stress.

Here, we have investigated the physical and molecular basis of stability of Arabidopsis DNA Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity stress in connection with the function of the N-terminal BRCT (breast cancer-associated C terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein. Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments using purified recombinant full length Pol λ and its N-terminal deletion forms have revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the other hand, the highly conserved C-terminal catalytic core PolX domain maintained its tertiary folds under similar condition. Circular dichroism (CD) and fourier transform infrared (FT-IR) spectral studies have indicated appreciable change in the secondary structural elements in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration. In vivo aggregation studies using transient expression systems in Arabidopsis and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunoprecipitation assays revealed interaction of Pol λ with the eukaryotic molecular chaperone HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell. Overall, our results have provided one of the first comprehensive information on the biophysical characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro rich modules in regulating the stability of this protein under genotoxic stress in plants.

Comparison of physico-chemical aspects between E. coli and human dihydrofolate reductase: an equilibrium unfolding study

A protein, differing in origin, may exhibit variable physicochemical behaviour, difference in sequence homology, fold and function. Thus studying structure-function relationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function relationship. Dihydrofolate reductase is an enzyme involved in cell cycle regulation. It is a significant enzyme as.a target for developing anticancer drugs. Hence, detailed understanding of structure-function relationships of wide variants of the enzyme dihydrofolate reductase would be important for developing an inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In this communication, we have reported the comparative structure-function relationship between E. coli and human dihydrofolate reductase. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties of these two proteins like relative' stability differences and variation in conformational changes under identical denaturing conditions. The equilibrium unfolding mechanism of dihydrofolate reductase proteins using guanidine hydrochloride as a denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, intrinsic tryptophan fluorescence and an extrinsic fluorophore 8-anilino-1-naphthalene-sulfonic acid as probes. It has been observed that osmolytes, such as 1M sucrose, and 30% glycerol, provided enhanced stability to both variants of dihydrofolate reductase. Their level of stabilisation has been observed to be dependent on intrinsic protein stability. It was observed that 100 mM proline does not show any 'significant stabilisation to either of dihydrofolate reductases. In the present study, it has been observed that the human protein is relatively less stable than the E.coli counterpart.

The Tale of Two Domains

Two-partner secretion (TPS) systems, harboring both A and B-components, are present in nearly all gram-negative bacteria. TpsB components selectively secrete their cognate TpsA exoprotein in an energy independent and vectorial manner across the outer membrane. Upon secretion, TpsA proteins function as adhesins, cytolysins, and heme binding proteins. In order to more fully understand the structure and folding of the TpsA protein family, a truncated form of hemolysin (HpmA265) was investigated. HpmA265 harbors a right-handed, parallel β-helix structure that is topped and flanked via segments of anti-parallel β-sheet. Previous structural studies have suggested a variety means to stabilize the β-helix structure including inner core electrostatic interactions and an inner core disulfide bond shared between cysteine 144 and 147. Equilibrium unfolding studies were implemented to investigate the impact of replacing glutamine 125 and the two cysteine residues on the stability of the HpmA265 β-helix structure. Multi-transitional denaturation results reported within this study provide evidence that the β-helix is comprised of two distinct and thermodynamically independent domains, termed the amino cap and β-helix core. The ΔG°H2O for folding the amino-cap is much larger than the β-helix core, implicating the buried and insulated hydrogen bonds as a primary means to stabilize the domain. Functionally, we propose the free-energy contribution from the stability within the amino-cap provides substantial selectivity and stability during the energy independent and vectorial folding of two-partner secretion pathway family members. This work was supported, in whole or in part, by National Science Foundation Grants MCB1050435 (to TMW) and MCB1434473 (to MRW), and a University Wisconsin – La Crosse Faculty Research Grant (to TMW).

Collapse of a long axis: single-molecule Förster resonance energy transfer and serpin equilibrium unfolding.

The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations. This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.

Statistical Mechanical Models for Analyzing the Site-Specific Folding of helix-turn-helix Motifs

Isotopically-edited IR spectroscopy can provide detailed site-specific information about the protein folding mechanism. With our equilibrium unfolding studies of two simple helix-turn-helix (hth) proteins using circular dichroism and 13C isotopically labeled infrared spectroscopy, we incorporated this experimental data into Ising-like statistical mechanical models to better understand the observed structural and energetic properties of the two proteins. Two variations of the Ising-like model were implemented: the Wato-Saito-Muñoz-Eaton (WSME) model, which can be enumerated exactly using efficient transfer matrix methods, and the Baker-Finkelstein (BF) model using a double-sequence approximation. Model parameters were optimized by simultaneously fitting the complete set of data for the whole protein as well as each helix independently to reflect what was observed through experiments. In order to give a more realistic representation of protein energy, various statistical residue-specific potential matrices were tested as the inter-residue contact energy in the model. We found that different statistical potentials varied in its success to simultaneously fit all the experimental data, however all the residue-specific matrices resulted in an improvement over considering only a single parameter for the contact energy. Both the WSME and BF models were able to reproduce the equilibrium unfolding data when analyzing the hth proteins as a whole, but the WSME model could not correctly predict the folding of only the helix when analyzed independently due to the assumptions of the model. On the other hand, the BF model was capable of reproducing the experimental data for both the whole protein and the independent helices.

A Functional Loop Spanning Distant Domains of Glutaminyl-tRNA Synthetase Also Stabilizes a Molten Globule State

Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS.