Revisiting absorbance at 230 nm as a protein unfolding probe

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A₂₃₀) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A₂₃₀ as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A₂₃₀. Significant changes in A₂₃₀ were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A₂₃₀ in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A₂₃₀ under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A₂₃₀ allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A₂₃₀ is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.     

Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins

The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H⁺ binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pK_a values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pK_a values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pK_a values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.     

Structural Determinants for Improved Stability of Designed Ankyrin Repeat Proteins with a Redesigned C-Capping Module

Designed ankyrin repeat proteins (DARPins) that specifically bind to almost any target can be obtained by ribosome display or phage display from combinatorial libraries. Although DARPins are already very stable molecules, molecular dynamics simulations, equilibrium denaturation experiments, structural studies, and recent NMR experiments suggested that the unfolding of the original C-terminal capping repeat (C-cap), taken from a natural ankyrin repeat protein, limits the stability of the initial DARPin design. Several point mutations had been introduced to optimize the C-cap and were shown to indeed further increase the stability of DARPins. We now determined crystal structures of DARPins with one or three full-consensus internal repeats (NI₁C or NI₃C) between an N-terminal capping repeat and mutants of the C-cap. An NI₁C mutant, in which the C-cap was only extended by three additional helix-forming residues, showed no structural change but reduced B-factors in the C-cap. An NI₃C C-cap mutant carrying five additional mutations in the interface to the preceding repeat, previously designed by using the consensus sequence as a guide, showed a rigid-body movement of the C-cap towards the internal repeat. This movement results in an increased buried surface area and a superior surface complementarity and explains the improved stability in equilibrium unfolding, compared to the original C-cap. A C-cap mutant with three additional mutations introducing suitably spaced charged residues did not show formation of salt bridges, explaining why its stability was not increased further. These structural studies underline the importance of repeat coupling for stability and help in the further design of this protein family.     

Effects of subclass change on the structural stability of chimeric,humanized, and human antibodies under thermal stress

To address how changes in the subclass of antibody molecules affect their thermodynamic stability, we prepared three types of four monoclonal antibody molecules (chimeric, humanized, and human) and analyzed their structural stability under thermal stress by using size‐exclusion chromatography, differential scanning calorimetry (DSC), circular dichroism (CD), and differential scanning fluoroscopy (DSF) with SYPRO Orange as a dye probe. All four molecules showed the same trend in change of structural stability; the order of the total amount of aggregates was IgG1 < IgG2 < IgG4. We thus successfully cross‐validated the effects of subclass change on the structural stability of antibodies under thermal stress by using four methods. The T_h values obtained with DSF were well correlated with the onset temperatures obtained with DSC and CD, suggesting that structural perturbation of the CH2 region could be monitored by using DSF. Our results suggested that variable domains dominated changes in structural stability and that the physicochemical properties of the constant regions of IgG were not altered, regardless of the variable regions fused.     

Kinetic Control in Protein Folding for Light Chain Amyloidosis and the Differential Effects of Somatic Mutations

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis–trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans–cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.     

An effective thiol-reactive probe for differential scanning fluorimetry with a standard real-time polymerase chain reaction device

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the K_d values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT–PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL l-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT–PCR machines in a hydrophilic or hydrophobic environment.     

Fold-Unfold Transitions in the Selectivity and Mechanism of Action of the N-Terminal Fragment of the Bactericidal/Permeability-Increasing Protein (rBPI21)

Septic or endotoxic shock is a common cause of death in hospital intensive care units. In the last decade numerous antimicrobial peptides and proteins have been tested in the search for an efficient drug to treat this lethal disease. Now in phase III clinical trials, rBPI₂₁, a recombinant N-terminal fragment of the bactericidal/permeability-increasing protein (BPI), is a promising drug to reduce lesions caused by meningococcal sepsis. We correlated structural and stability data with functional information of rBPI₂₁ bound to both model systems of eukaryotic and bacterial membranes. On interaction with membranes, rBPI₂₁ loses its conformational stability, as studied by circular dichroism. This interaction of rBPI₂₁ at membrane level was higher in the presence of negatively charged phospholipid relatively to neutral ones, with higher partition coefficients (K_p), suggesting a preference for bacterial membranes over mammalian membranes. rBPI₂₁ binding to membranes is reinforced when its disulfide bond is broken due to conformational changes of the protein. This interaction is followed by liposome aggregation due to unfolding, which ensures protein aggregation, and interfacial localization of rBPI₂₁ in membranes, as studied by extensive quenching by acrylamide and 5-deoxylstearic acid and not by 16-deoxylstearic acid. An uncommon model of the selectivity and mechanism of action is proposed, where membrane induces unfolding of the antimicrobial protein, rBPI₂₁. The unfolding ensures protein aggregation, established by protein-protein interaction at membrane surface or between adjacent membranes covered by the unfolded protein. This protein aggregation step may lead to membrane perturbation.     

Investigating protein unfolding kinetics by pulse proteolysis

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (K_d) of the complex from unfolding kinetic constants, which is also in good agreement with known K_d values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.     

Direct and simple detection of recombinant proteins from cell lysates using differential scanning fluorimetry

A simple, inexpensive, and universal method to quantify the recombinant proteins in Escherichia coli cell lysate using differential scanning fluorimetry (DSF) is reported. This method is based on the precise correlation between Δ(fluorescence intensity) determined by DSF and the amount of protein in solution. We first demonstrated the effectiveness of the DSF method using two commercially available enzymes, α-amylase and cellobiase, and then confirmed its utility with two recombinant proteins, amylosucrase and maltogenic amylase, expressed in E. coli. The Δ(fluorescence intensity) in DSF analysis accurately correlated with the concentration of the purified enzymes as well as the recombinant proteins in E. coli cell lysates. The main advantage of this method over other techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and green fluorescence protein (GFP) fusion proteins is that intact recombinant protein can be quantified without the requirement of additional chemicals or modifications of the recombinant protein. This DSF assay can be performed using widely available equipment such as a real-time polymerase chain reaction (RT–PCR) instrument, microplates or microtubes, and fluorescent dye. This simple but powerful method can be easily applied in a wide range of research areas that require quantification of expressed recombinant proteins.     

Unfolding of CBP21, a lytic polysaccharide monooxygenase,without dissociation of its copper ion cofactor

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu²⁺ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t_p, for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of t_p in the presence of excess Cu²⁺ was around 75°C, indicating that Cu²⁺ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu²⁺ ⇌ U∙Cu²⁺, where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol⁻¹ in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu²⁺ complex itself.     

The foldon substructure of staphylococcal nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔG_HX) and the kinetic unfolding and refolding rates (k_op and k_cl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 × 10^− 6 s^− 1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.     

Intrinsic affinity of protein – ligand binding by differential scanning calorimetry

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.     

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P₁ (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P′₂ favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P₁ and P′₂ substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.     

A Comprehensive Alanine-Scanning Mutagenesis Study Reveals Roles for Salt Bridges in the Structure and Activity of Pseudomonas aeruginosa Elastase

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k _cat/K _m), half-lives (t _1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.     

Probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis

Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of ∼16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm_app) by 1–6°C resulting in an increase in free energy of unfolding (ΔG_unf) by 0.04–1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with ΔG_unf, (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (ΔΔG_unf = 1.57 kcal⁻¹) justifies the robustness of this relation.     

Substitution of carbonate by non-physiological synergistic anion modulates the stability and iron release kinetics of serum transferrin

Serum transferrin (sTf) is a bi-lobal protein. Each lobe of sTf binds one Fe³⁺ ion in the presence of a synergistic anion. Physiologically, carbonate is the main synergistic anion but other anions such as oxalate, malonate, glycolate, maleate, glycine, etc. can substitute for carbonate in vitro. The present work provides the possible pathways by which the substitution of carbonate with oxalate affects the structural, kinetic, thermodynamic, and functional properties of blood plasma sTf. Analysis of equilibrium experiments measuring iron release and structural unfolding of carbonate and oxalate bound diferric-sTf (Fe₂sTf) as a function of pH, urea concentration, and temperature reveal that the structural and iron-centers stability of Fe₂sTf increase by substitution of carbonate with oxalate. Analysis of isothermal titration calorimetry (ITC) scans showed that the affinity of Fe³⁺ with apo-sTf is enhanced by substituting carbonate with oxalate. Analysis of kinetic and thermodynamic parameters measured for the iron release from the carbonate and oxalate bound monoferric-N-lobe of sTf (Fe_NsTf) and Fe₂sTf at pH 7.4 and pH 5.6 reveals that the substitution of carbonate with oxalate inhibits/retards the iron release via increasing the enthalpic barriers.     

Engineered Bi-Histidine Metal Chelation Sites Map the Structure of the Mechanical Unfolding Transition State of an Elastomeric Protein Domain GB1

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The ϕ^M2+_U-value is defined as ΔΔG_‡-N/ΔΔG_U-N, which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG_‡-N) relative to its thermodynamic stability (ΔG_U-N) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify ϕ^M2+_U-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1’s mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.     

Overview — In vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC₅₀=36.4 μM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC₅₀=4.62 μM). We isolate and identify a carbamoylated peptide at Cys₃₀₂ with the sequence FNQGQC₃₀₁C₃₀₂C₃₀₃. Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys₃₀₂, present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.     

Influence of the stability of a fused protein and its distance to the amyloidogenic segment on fibril formation

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔG _D = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔG _D = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.     

Order Statistics Theory of Unfolding of Multimeric Proteins

Dynamic force spectroscopy has become indispensable for the exploration of the mechanical properties of proteins. In force-ramp experiments, performed by utilizing a time-dependent pulling force, the peak forces for unfolding transitions in a multimeric protein (D)_N are used to map the free energy landscape for unfolding for a protein domain D. We show that theoretical modeling of unfolding transitions based on combining the observed first (f₁), second (f₂), …, N^th (f_N) unfolding forces for a protein tandem of fixed length N, and pooling the force data for tandems of different length, n₁ < n₂ < … < N, leads to an inaccurate estimation of the distribution of unfolding forces for the protein D, ψ_D(f). This problem can be overcome by using Order statistics theory, which, in conjunction with analytically tractable models, can be used to resolve the molecular characteristics that determine the unfolding micromechanics. We present a simple method of estimation of the parent distribution, ψ_D(f), based on analyzing the force data for a tandem (D)_n of arbitrary length n. Order statistics theory is exemplified through a detailed analysis and modeling of the unfolding forces obtained from pulling simulations of the monomer and oligomers of the all-β-sheet WW domain.