Presence Of GdmCl Research Articles

Molecular level insight into the counteraction of trehalose on the activity as well as denaturation of lysozyme induced by guanidinium chloride

The effect of trehalose on the activity and structural change of lysozyme induced by the chemical denaturant guanidinium chloride (GdmCl) has been investigated using the activity assay, spectroscopy as well as molecular dynamics simulation methods. The activity along with secondary and tertiary content of lysozyme decreases drastically in the presence of GdmCl whereas the addition of trehalose in GdmCl reverses the effect. It has been observed that the addition of trehalose in GdmCl affects mainly to the active site of lysozyme unlike the case of urea in which water gets accommodate at the surface of the protein. This study suggests that addition of trehalose in GdmCl changes the solvent environment more like to the native condition in the active site of lysozyme and modifies the electrostatic and Lennard-Jones interactions which play an important role in the reversal of its activity as well as structural change.

Estimation of structure and stability of MurE ligase from Salmonella enterica serovar Typhi

MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔGD0 = 3.13 kcal mol−1, m = 1.52 kcal mol−1 M−1 and Cm (=ΔGD0/m) = 2.05 M were calculated in the presence of GdmCl whereas in the case of urea these were ΔGD0 = 3.04 kcal mol−1, m = 1.20 kcal mol−1 M−1 and Cm (=ΔGD0/m) = 2.53 M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.

Mutational probing of protein aggregates to design aggregation-resistant proteins.

Characterization of amorphous protein aggregates may offer insights into the process of aggregation. Eleven single amino acid mutants of lipase (LipA of Bacillus subtilis) were subjected to temperature‐induced aggregation, and the resultant aggregates were characterized for recovery of activity in the presence of guanidinium chloride (GdmCl). Based on activity recovery profiles of the aggregates, the mutants could be broadly assigned into four groups. By including at least one mutation from each group, a mutant was generated that showed an increase of ~ 10 °C in melting temperature (T m) compared to the wild‐type and did not aggregate even at 75 °C. This method explores characterization of amorphous protein aggregates in the presence of GdmCl and helps in identifying mutations involved in protein aggregation.

Collective hydration dynamics of guanidinium chloride solutions and its possible role in protein denaturation: a terahertz spectroscopic study.

The remarkable ability of guanidinium chloride (GdmCl) to denature proteins is a well studied yet controversial phenomenon; the exact molecular mechanism is still debatable, especially the role of hydration dynamics, which has been paid less attention. In the present contribution, we have addressed the issue of whether the collective hydrogen bond dynamics of water gets perturbed in the presence of GdmCl and its possible impact on the denaturation of a globular protein human serum albumin (HSA), using terahertz (THz) time domain spectroscopy (TTDS) in the frequency range of 0.3-2.0 THz. The collective hydrogen bond dynamics is determined by fitting the obtained complex dielectric response in a multiple Debye relaxation model. To compare the results, the studies were extended to two more salts: tetramethylguanidinium chloride (TMGdmCl) and sodium chloride (NaCl). It was concluded that the change in hydration dynamics plays a definite role in the protein denaturation process.

Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate

A subtilisin E variant (M4) showing high activity and resistance towards guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS) was previously identified after three rounds of directed evolution [Li et al., ChemBioChem 2012, 13(5), 691–699.]. In this report, 10 additional positions, identified during directed subtilisin E evolution, were saturated on the previously reported SeSaM1-5 variant (S62/A153/G166/I205). Screening confirmed that chaotolerant variants included amino acid substitutions either in the active site, or the substrate binding pocket. Two variants, M5 (S62I/A153V/G166S/T224A/T240S) and M6 (S62I/A153V/G166S/I205V/N218S/T224A) were finally generated to maximize activity and stability in the presence of GdmCl or SDS. The inactivation concentration (IC50) of M6 using Suc-AAPF-pNA as substrate was significantly increased compared to M4 in the presence of GdmCl (IC50 (M4): 2.7M; IC50 (M6): 4.6M) and SDS (IC50 (M4): 1.5%; IC50 (M6): 4.0%). The half-life in 5M GdmCl was also significantly improved for M6 compared to M4 (t 1/2 (M4): 2min; t 1/2 (M6): 15min). M5 retained resistance towards GdmCl or SDS as in M4. The activity of M5 towards a complex protein substrate (Azocasein) was increased by ∼1.5 fold compared to M4 and M6. Circular dichroism (CD) analysis for subtilisin E wild type (WT) and three variants (M4, M5 and M6) indicated that secondary structures of all variants including wild type at 1–2M GdmCl (except M4) were not significantly perturbed, with unfolding occurring for WT and all three variants above 3M GdmCl. In SDS, the secondary structures of WT and all three variants remained intact at concentrations of 0.5 to 2.0% (w/v) SDS. Results suggest that subtilisin E inactivation occurred most likely due to inhibitory effect, since a general unfolding of the enzyme was not observed through circular dichroism. Such inhibition could be avoided by limiting the access of GdmCl and SDS to the active site and/or to residues involved in substrate binding.

The Molecular Mechanism of Hsp100 Chaperone Inhibition by the Prion Curing Agent Guanidinium Chloride

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called "prion curing," the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm(+) ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm(+) interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones.

Effects of Hofmeister Ions on the α-Helical Structure of Proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO4 in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO4 originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO4 denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state.

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41kDa monomer. The enzyme showed Km values of 41μM and 34μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with Ki value of 0.5μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339nm to 357nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.

Structural Analysis of a Periplasmic Binding Protein in the Tripartite ATP-independent Transporter Family Reveals a Tetrameric Assembly That May Have a Role in Ligand Transport

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Critical role of tryptophanyl residues in the conformational stability of goose δ-crystallin

δ-Crystallin is the major structural protein in avian and reptilian eye lenses but its sequence is highly homologous with the urea cycle enzyme, argininosuccinate lyase (ASL). In previous studies the multi-step unfolding process of this protein in the presence of GdmCl was sensitively probed using tryptophan fluorescence. In this study the contribution of single tryptophan residues to the stability of the local environment was monitored by mutation of two highly conservative tryptophan residues in goose δ-crystallin, Trp 74 and Trp 169. These residues behaved differently in terms of fluorescence intensity and maxima emission wavelength, consistent with their structural location in buried or solvent accessible regions. No gross changes in the secondary structure after mutation were observed, as judged by far-UV CD. The side chains of tryptophan residues in the structure of wild-type goose δ-crystallin possess both hydrophobic and hydrogen bonding interactions. Replacement of the side chain with phenylalanine or alanine led to expose of a hydrophobic area and a reduction in thermal stability; W169A particularly has a T m value that was 10 °C lower than the wild type enzyme. In the presence of GdmCl, a sharp red shift in fluorescence wavelength due to subunit dissociation can be sensitively detected using a single tryptophan, with the region surrounding W74 undergoing the first transition with a [GdmCl] 1/2 of 0.45 M. Further measurement of unfolding curves by CD revealed that the W169A mutant was most unstable with a [GdmCl] 1/2 of 0.22 M. From sedimentation velocity analysis, the unstable conformation of the W169A mutant affected the assembly of the quaternary structure. Our studies demonstrate the critical role for the tryptophan residues in stabilizing protein conformations and subunit assembly in δ-crystallin.

The effect of N-terminal truncation on double-dimer assembly of goose δ-crystallin

Delta-crystallin is a soluble structural protein in avian eye lenses that confers special refractive properties. In the presence of GdmCl (guanidinium chloride), tetrameric delta-crystallin undergoes dissociation via a dimeric state to a monomeric molten globule intermediate state. The latter are denatured at higher GdmCl concentrations in a multi-state manner. In the present study, the X-ray structure of goose delta-crystallin was determined to 2.8 A (1 A=0.1 nm). In this structure the first 25 N-terminal residues interact with a hydrophobic cavity in a neighbouring molecule, stabilizing the quaternary structure of this protein. When these 25 residues were deleted this did not produce any gross structural changes, as judged by CD analysis, but slightly altered tryptophan fluorescence and ANS (8-anilino-1-naphthalenesulphonic acid) spectra. The dimeric form was significantly identified as judged by sedimentation velocity and nondenaturing gradient gel electrophoresis. This mutant had increased sensitivity to temperature denaturation and GdmCl concentrations of 0.3-1.0 M. This protein was destabilized about 3.3 kcal/mol (1 kcal=4.184 kJ) due to N-terminal truncation. After incubation at 37 degrees C N-terminal truncated proteins were prone to aggregation, suggesting the presence of the unstable dimeric conformation. An important role for the N-terminus in dimer assembly of goose delta-crystallin is proposed.

Conformational Study of Spectrin in Presence of Submolar Concentrations of Denaturants

The presence of very low concentrations of the commonly used chemical denaturants, guanidinium chloride (GdmCl) and urea brought about conformational changes in the erythrocyte membrane skeletal protein, spectrin. Evidences in support of changes in the quaternary structure of spectrin have been put forward from quenching study of tryptophan fluorescence, by both steady state and time-resolved measurements, using acrylamide as the quencher. It revealed significant differences between the Stern-Volmer quenching constants (K(SV)) and the fraction of accessible tryptophans (f(e)) observed in absence and presence of GdmCl and urea concentrations below 1 M at which the association of the two subunits remains intact. The steady state anisotropy of both the spectrin tryptophans and the spectrin-bound fluorescence probe, Prodan also indicate changes in the overall flexibility of the spectrin dimer, originating from changes in the quaternary structure of spectrin. Studies on the binding of Prodan, further indicate that conformational changes also occur in spectrin near the Prodan-binding site at the terminal domain of the protein which is reflected in 3-4 fold decrease in the affinity of binding of Prodan to spectrin in the presence of GdmCl and urea compared to that observed in the absence of the denaturants. The dissociation constant (K(d)) of Prodan to spectrin is 0.43 microM at 25 degrees C.

Guanidinium chloride denaturation of the dimeric Bacillus licheniformis BlaI repressor highlights an independent domain unfolding pathway.

The Bacillus licheniformis 749/I BlaI repressor is a prokaryotic regulator that, in the absence of a beta-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase. The BlaI repressor is composed of two structural domains. The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain. Recent studies have shown the existence of the monomeric, dimeric and tetrameric forms of BlaI in solution. In the present study, we analyse the equilibrium unfolding of BlaI in the presence of GdmCl (guanidinium chloride) using different techniques: intrinsic and ANS (8-anilinonaphthalene-l-sulphonic acid) fluorescence, far- and near-UV CD spectroscopy, cross-linking, analytical ultracentrifugation, size exclusion chromatography and NMR spectroscopy. In addition, the intact NTD and CTD were purified after proteolysis of BlaI by papain, and their unfolding by GdmCl was also studied. GdmCl-induced equilibrium unfolding was shown to be fully reversible for BlaI and for the two isolated fragments. The results demonstrate that the NTD and CTD of BlaI fold/unfold independently in a four-step process, with no significant co-operative interactions between them. During the first step, the unfolding of the BlaI CTD occurs, followed in the second step by the formation of an 'ANS-bound' intermediate state. Cross-linking and analytical ultracentrifugation experiments suggest that the dissociation of the dimer into two partially unfolded monomers takes place in the third step. Finally, the unfolding of the BlaI NTD occurs at a GdmCl concentration of approx. 4 M. In summary, it is shown that the BlaI CTD is structured, more flexible and less stable than the NTD upon GdmCl denaturation. These results contribute to the characterization of the BlaI dimerization domain (i.e. CTD) involved in the induction process.

Anion binding to the ubiquitin molecule.

Effects of different salts (NaCl, MgCl2, CaCl2, GdmCl, NaBr, NaClO4, NaH2PO4, Na2SO4) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for Cl- show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of -15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01-2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of Cl- ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with Cl- binding effects.