Effect of amino acid substitutions at the subunit interface on the stabiity and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.

Chemical unfolding of chicken villin headpiece in aqueous dimethyl sulfoxide solution: cosolvent concentration dependence, pathway, and microscopic mechanism

Unfolding of a protein often proceeds through partial unfolded intermediate states (PUIS). PUIS have been detected in several experimental and simulation studies. However, complete analyses of transitions between different PUIS and the unfolding trajectory are sparse. To understand such dynamical processes, we study chemical unfolding of a small protein, chicken villin head piece (HP-36), in aqueous dimethyl sulfoxide (DMSO) solution. We carry out molecular dynamics simulations at various solution compositions under ambient conditions. In each concentration, the initial step of unfolding involves separation of two adjacent native contacts, between phenyl alanine residues (11-18 and 7-18). This first step induces, under appropriate conditions, subsequent separation among other hydrophobic contacts, signifying a high degree of cooperativity in the unfolding process. The observed sequence of structural changes in HP-36 on increasing DMSO concentration and the observed sequence of PUIS, are in approximate agreement with earlier simulation results (in pure water) and experimental observations on unfolding of HP-36. Peculiar to water-DMSO mixture, an intervening structural transformation (around 15% of DMSO) in the binary mixture solvent retards the progression of unfolding as composition is increased. This is reflected in a remarkable nonmonotonic composition dependence of RMSD, radius of gyration and the fraction of native contacts. At 30% mole fraction of DMSO, we find the extended randomly coiled structure of the unfolded protein. The molecular mechanism of DMSO induced unfolding process is attributed to the initial preferential solvation of the hydrophobic side chain atoms through the methyl groups of DMSO, followed by the hydrogen bonding of the oxygen atom of DMSO to the exposed backbone NH groups of HP-36.

Composition dependent multiple structural transformations of myoglobin in aqueous ethanol solution: A combined experimental and theoretical study

Experimental studies (circular dichroism and ultra-violet (UV) absorption spectra) and large scale atomistic molecular dynamics simulations (accompanied by order parameter analyses) are combined to establish a number of remarkable (and unforeseen) structural transformations of protein myoglobin in aqueous ethanol mixture at various ethanol concentrations. The following results are particularly striking. (1) Two well-defined structural regimes, one at x(EtOH) similar to 0.05 and the other at x(EtOH) similar to 0.25, characterized by formation of distinct partially folded conformations and separated by a unique partially unfolded intermediate state at x(EtOH) similar to 0.15, are identified. (2) Existence of non-monotonic composition dependence of (i) radius of gyration, (ii) long range contact order, (iii) residue specific solvent accessible surface area of tryptophan, and (iv) circular dichroism spectra and UV-absorption peaks are observed. Interestingly at x(EtOH) similar to 0.15, time averaged value of the contact order parameter of the protein reaches a minimum, implying that this conformational state can be identified as a molten globule state. Multiple structural transformations well known in water-ethanol binary mixture appear to have considerably stronger effects on conformation and dynamics of the protein. We compare the present results with studies in water-dimethyl sulfoxide mixture where also distinct structural transformations are observed along with variation of co-solvent composition. (C) 2015 AIP Publishing LLC.

Differential effects of TR ligands on hormone dissociation rates: Evidence for multiple ligand entry/exit pathways

Some nuclear receptor (NR) ligands promote dissociation of radiolabeled bound hormone from the buried ligand binding cavity (LBC) more rapidly than excess unlabeled hormone itself This result was interpreted to mean that challenger ligands bind allosteric sites on the LBD to induce hormone dissociation, and recent findings indicate that ligands bind weakly to multiple sites on the LBD surface. Here we show, that a large fraction of thyroid hormone receptor (TR) ligands promote rapid dissociation (T(1/2) < 2 h) of , radiolabeled T(3) vs. T(3) (T(1/2), approximate to 5-7 h). We cannot discern relationships between this effect and ligand size, activity or affinity for TR beta. One ligand, GC-24, binds the TR LBC and (weakly) to the TR beta-LBD surface that mediates dimer/heterodimer interaction, but we cannot link this interaction to rapid T(3) dissociation. Instead, several lines of evidence suggest that the challenger ligand must interact with the buried LBC to promote rapid T(3) release. Since previous molecular dynamics simulations suggest that TR ligands leave the LBC by several routes, we propose that a subset of challenger ligands binds and stabilizes a partially unfolded intermediate state of TR that arises during T(3) release and that this effect enhances hormone dissociation. (C) 2009 Elsevier Ltd. All rights reserved.

The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTR At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease. (C) 2008 Elsevier B.V. All rights reserved.

Tertiary structural changes of the alpha-hemolysin from Staphylococcus aureus on association with liposome membranes

The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane, the second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition. (C) 1998 Academic Press.

SecB binds only to a late native-like intermediate in the folding pathway of barstar and not to the unfolded state

SecB is a cytosolic, tetrameric chaperone of Escherichia coli which maintains precursor proteins in a translocation competent state. We have investigated the effect of SecB on the refolding kinetics of the small protein barstar in I M guanidine hydrochloride at pH 7.0 and 25 degrees C using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to a late near-native intermediate along the folding pathway. For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models.

Probing conformational changes in orphan nuclear receptor: The NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the a-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners. (C) 2008 Elsevier B.V. All rights reserved.

Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate

On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (i) truncate hydrophobic side chains in native helix packing sites, (ii) truncate hydrophobic side chains not involved in interhelical contacts, and (iii) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate.

Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway.

Apomyoglobin folding proceeds through a molten globule intermediate (low-salt form; I1) that has been characterized by equilibrium (pH 4) and kinetic (pH 6) folding experiments. Of the eight alpha-helices in myoglobin, three (A, G, and H) are structured in I1, while the rest appear to be unfolded. Here we report on the structure and stability of a second intermediate, the trichloroacetate form of the molten globule intermediate (I2), which is induced either from the acid-unfolded protein or from I1 by > or = 5 mM sodium trichloroacetate. Circular dichroism measurements monitoring urea- and acid-induced unfolding indicate that I2 is more highly structured and more stable than I1. Although I2 exhibits properties closer to those of the native protein, one-dimensional NMR spectra show that it maintains the lack of fixed side-chain structure that is the hallmark of a molten globule. Amide proton exchange and 1H-15N two-dimensional NMR experiments are used to identify the source of the extra helicity observed in I2. The results reveal that the existing A, G, and H helices present in I1 have become more stable in I2 and that a fourth helix--the B helix--has been incorporated into the molten globule. Available evidence is consistent with I2 being an on-pathway intermediate. The data support the view that apomyoglobin folds in a sequential fashion through a single pathway populated by intermediates of increasing structure and stability.

Knots in rings - The circular knotted protein momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys(1)-Cys(18) disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy--D-glucose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from D-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.