Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.     

Circular dichroism study on the early folding events of beta-lactoglobulin entrapped in wet silica gels

beta-Lactoglobulin is a predominantly beta-sheet protein that folds by forming excess alpha-helices within milliseconds. In this study, the refolding of beta-lactoglobulin was dramatically decelerated by entrapping in wet nanoporous silica gel matrices, and monitored on a time scale of minutes or hours by far-UV circular dichroism spectroscopy. Analysis of kinetics and transient spectra allowed to define the sequence of folding events that consist of alpha-helical formation, beta-sheet core formation, and alpha-to-beta transition. The results suggest that the initially formed alpha-helices, presumably including the native alpha-helix, help to guide the formation of the adjacent beta-sheet core.     

Secondary structure in ribonuclease. II. Relations between folding kinetics and secondary structure elements

The kinetics of regain of 2′-CMP binding are monitored during renaturation of RNAase S. Experiments were performed by mixing equimolar amounts of S-peptide with S-protein. The S-protein fragment was incubated initially (i.e. before mixing with S-peptide) at pH 6.2 or 1.7 and various guanidine hydrochloride (GuHCl) concentrations. Three well-resolved phases are observed. The fastest phase is second-order. The reciprocal half-time increases linearly with fragment concentration and is independent of initial conditions for the S-protein fragment. An apparent on rate of k_on = 2 × 10⁵m^?1s^?1 is measured in 0.5 m-GuHCl (pH 6.2) and 20 ° C. Identical association kinetics are observed by changes in tyrosine absorbance. The fraction of native RNAase S formed in this second-order reaction strictly equals the fraction of S-protein molecules with intact β-sheet in initial conditions. The relation holds for different pH values, GuHCl concentrations and temperatures. The fraction of apparent helical content of S-protein in initial conditions may also vary but this is not reflected by the association reaction. We interpret this to mean that the β-sheet but not the α-helices must be preformed in initial conditions in order to generate the high-affinity peptide binding site of S-protein. Furthermore, it is concluded that the S-protein moiety β-sheet forms or unfolds in a single one-step reaction. 2′-CMP binding reports, additionally, two slower phases of renaturation. These are produced by S-protein molecules that have their β-sheet unfolded in initial conditions. It is observed that a unique dependence of these two folding rates exists for RNAase A, RNAase S and S-protein as function of t_m, the temperature of half-completion of thermal denaturation as measured by unfolding of the β-sheet in the respective compound in final conditions. The t_m value varies with changing pH, with GuHCl concentration and (for RNAase S) with changing fragment concentration. The findings are interpreted to argue in favor of a sequential mechanism of folding, where the stability of a structural precursor determines the rate of folding.     

Antigen Retrieval Causes Protein Unfolding: Evidence for a Linear Epitope Model of Recovered Immunoreactivity

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding.     

Evidence for the existence of an unfolding intermediate of thyroglobulin during denaturation by guanidine hydrochloride

The unfolding of bovine thyroglobulin (Tg) in guanidine hydrochloride (GuHCl) solution was studied by following the fluorescence and circular dichroism. With increasing GuHCl concentrations, the emission maximum of the intrinsic fluorescence clearly red-shifted in two stages. At concentrations of GuHCl less than 1.2 M or more than 1.6 M, the red shift showed a cooperative manner. At concentrations of GuHCl between 1.2 and 1.6 M, an unfolding intermediate was observed. It was further characterized by the increased binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid (ANS). No significant changes of the secondary structure were indicated by CD spectra at the concentrations of GuHCl between 1.2 and 1.6 M. The conformation of this state has properties similar to those of a molten globule state which may exist in the folding pathway of the protein. Further changes in fluorescence properties occurred at concentrations of denaturant higher than 1.6 M with a significant red shift of the emission maximum from 340 to 347 nm and a marked decrease in ANS binding. This in vitro study gave a clue to understand the biochemical mechanism for the occurrence of aggregation and molecular chaperone binding during Tg maturation in vivo.     

Structural and energetic differences between insertions and substitutions in staphylococcal nuclease.

In a previous study, the small protein staphylococcal nuclease was shown to readily accommodate single alanine and glycine insertions, with average losses in stability comparable to substitutions at the same sites (PROT. 7:299-305, 1990). To more fully explore this unexpected adaptability to changes in residue spacing, 2 double amino acid insertions (alanyl-glycine, glycyl-glycine) and 3 additional single amino acid insertions with dissimilar side chains (proline, leucine, and glutamine) were constructed at 10 of the sites previously studied. At 8 of these sites, the type of amino acid side chain on the inserted residue significantly influenced the stability of the mutant protein. However, at 9 of the 10 sites, the double insertions were found to be no more destabilizing than the single alanine or glycine insertions. In contrast, double substitution mutations of staphylococcal nuclease, which replace two adjacent residues with alanine, do not show this striking degree of non-additivity. A comparison of the effects of single glutamine and single glycine insertions with alanyl-glycine insertions indicates that insertion of alanine into the peptide backbone is, on average, less destabilizing than appending the equivalent atoms onto the side chain of a glycine insertion. To explain their very different energetic effects, we propose that, unlike most substitutions, the inserted residue(s) must induce lateral displacements of the polypeptide chain, forcing the folded conformation away from that of wild type. The resulting obligatory shifts in the positioning of residues flanking the insertion generate a large number of degrees of freedom around which the mutant structure can relax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the stability of fish light meromyosins by guanidine hydrochloride denaturation

The guanidine hydrochloride denaturation of light meromyosins (LMMs) of fish (carp, sardine and greenling) and rabbit was investigated to determine their structural stability quantitatively. The circular dichroism (CD) and fluorescence spectroscopies were applied to monitor denaturation. The CD results indicate that the LMM α-helix undergoes a two-step unfolding. The free energy of denaturation was calculated based on the linear extrapolation method and the denaturant binding model. Total free energies of the two-step unfolding of the α-helix are related to the water temperatures in which the fish live and the body temperature of rabbit. The stability of α-helical structure of LMM was in the following descending order: rabbit>carp>sardine>greenling. The free energies of denaturation obtained by tryptophan fluorescence differ from the free energies of the unfolding α-helix. The data from the two spectroscopic measurements are discussed along with the conformational changes of LMMs.     

Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping

Four residues Pro19, Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins, studied by means of CD measurements, proved reversible and well represented by the two-state ND transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.     

Comparison of Guanidine Hydrochloride (GdnHCl) and Urea Denaturation on Inactivation and Unfolding of Human Placental Cystatin (HPC)

The activity and conformational change of human placental cystatin (HPC), a low molecular weight thiol proteinase inhibitor (12,500) has been investigated in presence of guanidine hydrochloride (GdnHCl) and urea. The denaturation of HPC was followed by activity measurements, fluorescence spectroscopy and Circular Dichroism (CD) studies. Increasing the denaturant concentration significantly enhanced the inactivation and unfolding of HPC. The enzyme was 50% inactivated at 1.5 M GdnHCl or 3 M urea. Up to 1.5 M GdnHCl concentration there was quenching of fluorescence intensity compared to native form however at 2 M concentration intensity increased and emission maxima had 5 nm red shift with complete unfolding in 4–6 M range. The mid point of transition was in the region of 1.5–2 M. In case of urea denaturation, the fluorescence intensity increased gradually with increase in the concentration of denaturant. The protein unfolded completely in 6–8 M concentration of urea with a mid-point of transition at 3 M. CD spectroscopy shows that the ellipticity of HPC has increased compared to that of native up to 1.5 M GdnHCl and then there is gradual decrease in ellipticity from 2 to 5 M concentration. At 6 M GdnHCl the protein had random coil conformation. For urea the ellipticity decreases with increase in concentration showing a sigmoidal shaped transition curve with little change up to 1 M urea. The protein greatly loses its structure at 6 M urea and at 8 M it is a random coil. The urea induced denaturation follows two-state rule in which Native→Denatured state transition occurs in a single step whereas in case of GdnHCl, intermediates or non-native states are observed at lower concentrations of denaturant. These intermediate states are possibly due to stabilizing properties of guanidine cation (Gdn⁺) at lower concentrations, whereas at higher concentrations it acts as a classical denaturant.     

Multidomain structure of a recombinant streptokinase. A differential scanning calorimetry study

The temperature dependence of the heat capacity function of a recombinant streptokinase (rSK) has been studied by high-sensitivity differential scanning microcalorimetry and circular dichroism as a function of pH in low- and high-ionic strength buffers. At low ionic strength it is found that this protein, between pH 7 and 10, undergoes four reversible and independent two-state transitions during its unfolding, suggesting the existence of four domains in the native structure of the protein. This result reconciles previous conflicting reports about the number of domains of this protein obtained by differential scanning calorimetry and small-angle X-ray scattering. The number of two-state transitions decreases when the pH of the medium is decreased, without noticeable changes in its circular dichroism spectrum. A plausible localization of the four domains in the streptokinase sequences is proposed and their thermodynamic parameters are given. Increase of ionic strength to 200 mM NaCl affects positively the protein stability and confirms the existence of four reversible two-state transitions. Above 200 mM NaCl the protein stability decreases, resulting in low percentage of reversibility, and even irreversible transitions.     

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus

A [3Fe–4S]^1+/0 ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe–4S]^1+/0, with an unusually low reduction potential of ?650 mV, determined by cyclic voltammetry at pH 7.6. [¹H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe–4S]^1+/0 centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe–4S]^2+/1+ cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism.The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint (T_m) for thermally induced unfolding is 102±2 °C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 °C). The iron–sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.     

Agar Processed from Gracilaria foliifera of Florida

Agar producing a firm gel with specific rheological properties as well as a good commercial value was processed from Gracilaria foliifera (Forsskal) Børgesen, a red alga harvested along the Florida west coast. The alkali pretreatment of the seaweed based on the Funaki-Kojima’s method was applied before the process of agar extraction. The chemical and physical properties of the processed agar were compared with other typical agar experimentally processed.