Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0–3.0. Enzyme acquires partially unfolded conformation (U_P) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U_P state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.     

Biophysical characterization of fibroblast growth factor homologous factor-1b (FHF-1b): sodium dodecyl sulfate promotes two state folding

The current article describes the biophysical characterization and folding studies of fibroblast growth factor homologous factor-1b (FHF-1b) in comparison with acidic fibroblast growth factor (FGF-1). Our data indicates that FHF-1 is significantly more stable than FGF-1. The folding mechanism of these two proteins seems to be different although they share high degree of sequence and structural similarity. FHF-1 unfolds through stable intermediate state while unfolding of FGF-1 is two-state. Interestingly, low concentration of sodium dodecyl sulfate (SDS) drives the folding pathway of FHF-1b to two-state.     

Equilibrium unfolding of <Emphasis Type="Italic">A. niger</Emphasis> RNase: pH dependence of chemical and thermal denaturation

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A_MG1) into another (I_MG2).     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the alpha + beta class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0- 2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a beta-sheet conformation and shows a strong binding to 8-anilino-1- napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.     

Effects of deleting a tripeptide sequence observed in muscular dystrophy patients on the conformation of synthetic peptides corresponding to the scaffolding domain of caveolin-3

The caveolin-scaffolding domain (CSD) is a region in caveolin-1 and 3 that mediates interactions with signaling proteins. In some patients with limb-girdle muscular dystrophy, a three amino acid micro deletion in the CSD has been observed. The conformations and aggregation behavior of synthetic peptides, corresponding to the CSD of caveolin-3: DGVWKVSYTTFTVSKYWFY and the sequence where TFT (underlined in the native sequence) has been deleted, have been investigated. Circular dichroism spectra and molecular dynamics simulations indicate distinctive differences in the conformations of the native and mutant sequences. The extent of self-association in aqueous medium is also less pronounced in the case of the peptide with the micro deletion. It is likely that the structural changes arising as a result of TFT deletion distrupt oligomerization and consequently mistargeting and degradation.     

Single disulfide bond reduced papain exists in a compact intermediate state

Partially reduced proteins and other chemically modified derivatives are very useful model systems to understand the protein folding in vivo. Upon reduction, proteins attain different conformations with varying degrees of compactness. The reduction of papain in the presence of 8 M urea leads to the partial reduction of one disulfide bond. This derivative (single disulfide reduced carboxymethylated 1RCM papain (3RCM papain)) was characterized by spectroscopic methods and the effect of this reduction on the unfolding of the protein was investigated. Under this partial reduction, papain exhibits more than half of the tertiary and most of the secondary structures relative to the non-reduced molecule (free cysteine reduced and carboxymethylated papain (1RCM papain)). Hydrophobic regions are exposed to the solvent as observed through 8-anilino-1-naphthalene sulfonic acid binding which was absent in the fully intact and unfolded protein, at neutral pH. Hydrodynamic studies indicated that 3RCM papain, under neutral conditions, possess expanded conformation as compared to the native protein. Tryptophan fluorescence quenching studies suggested the exposure of aromatic residues to solvent. Guanidine hydrochloride induced unfolding of this derivative, at neutral pH, showed a non-cooperative transition contrary to the cooperativity seen with intact protein. Thermal unfolding indicates that 3RCM papain is less stable compared to the intact protein. These findings suggest that partial reduction of papain has a significant effect on the unfolding behavior of papain.     

Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na₂SO₄ to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.     

Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum

The major carotenoid pigments of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum, were identified as zeaxanthin, beta-cryptoxanthin, and beta-carotene. Analysis was based on ultraviolet-visible spectroscopy, mass spectroscopy, and reversed-phase HPLC. Photoacoustic spectroscopy of intact bacterial cells revealed that the bulk of the pigments in S. antarcticus and S. multivorum was associated with the cell membrane. In vitro studies with synthetic membranes of phosphatidylcholine demonstrated that the major pigment was bound to the membranes and decreased their fluidity. The relative amounts of polar pigments were higher in cells grown at 5 degrees C than in cells grown at 25 degrees C. In the mesophilic strain, the synthesis of polar carotenoids was quantitatively less than that of the psychrotolerant strain.