Equilibrium Studies of the Reaction of Turkey (Meleagris gallopavo) Haemoglobin Sulphydryl Groups with 5,5′-dithiobis(2-nitrobenzoate): Tertiary Conformational Change in Turkey Haemoglobin Induced by Inositol hexakisphosphate

2014 ◽  
Vol 4 (4) ◽  
pp. 200-206
Author(s):  
C. O. Ehi-Eromosele ◽  
A. Edobor-Osoh ◽  
C. O. Ajanaku ◽  
W. U. Anake ◽  
O. Aladesuyi ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Ph Dependence ◽  
Meleagris Gallopavo ◽  
Relative Distribution ◽  
Minor Components ◽  
Protein Conformations ◽  
Inositol Hexakisphosphate ◽  
Equilibrium Studies ◽  
Relative Population ◽  
Sulphydryl Groups

The red blood cell of turkey contains two haemoglobin types, major and minor components. In the present study, the equilibrium constant, Kequ, for the reaction of 5,5′-dithiobis(2-nitrobenzoate), DTNB, with the sulphydryl group of the major turkey aquomethaemoglobin was determined at 25°C as a function of pH. Kequ varies by about 2 to 3 orders of magnitude between pH 5.6 and 9.0 for both haemoglobin [stripped and in the presence of inositol hexakisphosphate (inositol-P6)]. Calculations from the pH dependence of Kequ showed that in the r ⇌ t tertiary conformational transition of aquomethae-moglobin, the t isomer population was 0.26 %. In the presence of inositol-P6, the t isomer population increased to 9.08 %. The results showed that while inositol-P6 increased the relative population of the t tertiary conformation by changing the relative distribution of two protein conformations, it had no effect on Kequ. The effect of Inositol-P6 on the nature and number of groups linked to the DTNB reaction was also determined.

scholarly journals The staphylococcal biofilm protein Aap forms a tetrameric species as a necessary intermediate before amyloidogenesis

2020 ◽  
Vol 295 (37) ◽  
pp. 12840-12850
Author(s):  
Alexander E. Yarawsky ◽  
Andrew B. Herr
Keyword(s):  
Staphylococcus Epidermidis ◽  
Analytical Ultracentrifugation ◽  
Conformational Transition ◽  
Mechanistic Model ◽  
Critical Factor ◽  
Equilibrium Studies ◽  
Dimer Interface ◽  
Biologically Relevant ◽  
Accumulation Associated Protein ◽  
Initial Assembly

The accumulation-associated protein (Aap) from Staphylococcus epidermidis is a biofilm-related protein that was found to be a critical factor for infection using a rat catheter model. The B-repeat superdomain of Aap, composed of 5–17 B-repeats, each containing a Zn2+-binding G5 and a spacer subdomain, is responsible for Zn2+-dependent assembly leading to accumulation of bacteria during biofilm formation. We previously demonstrated that a minimal B-repeat construct (Brpt1.5) forms an antiparallel dimer in the presence of 2–3 Zn2+ ions. More recently, we have reported the presence of functional amyloid-like fibrils composed of Aap within S. epidermidis biofilms and demonstrated that a biologically relevant construct containing five and a half B-repeats (Brpt5.5) forms amyloid-like fibrils similar to those observed in the biofilm. In this study, we analyze the initial assembly events of the Brpt5.5 construct. Analytical ultracentrifugation was utilized to determine hydrodynamic parameters of reversibly associating species and to perform linked equilibrium studies. Linkage studies indicated a mechanism of Zn2+-induced dimerization similar to smaller constructs; however, Brpt5.5 dimers could then undergo further Zn2+-induced assembly into a previously uncharacterized tetramer. This led us to search for potential Zn2+-binding sites outside of the dimer interface. We developed a Brpt5.5 mutant that was unable to form the tetramer and was concordantly incapable of amyloidogenesis. CD and dynamic light scattering indicate that a conformational transition in the tetramer species is a critical step preceding amyloidogenesis. This mechanistic model for B-repeat assembly and amyloidogenesis provides new avenues for potential therapeutic targeting of staphylococcal biofilms.

A pH-dependent conformational transition of Aβ peptide and physicochemical properties of the conformers in the glial cell

2002 ◽  
Vol 361 (3) ◽  
pp. 547-556 ◽  
Author(s):  
Yoichi MATSUNAGA ◽  
Nobuhiro SAITO ◽  
Akihiro FUJII ◽  
Junichi YOKOTANI ◽  
Tadakazu TAKAKURA ◽  
...  
Keyword(s):  
Glial Cell ◽  
Conformational Changes ◽  
Conformational Transition ◽  
Early Stage ◽  
Ph Dependence ◽  
Amyloid Β ◽  
Proteinase K ◽  
Aβ Peptide ◽  
Ph Dependent ◽  
Insight Into

In the present study we identified the epitopes of antibodies against amyloid β-(1–42)-peptide (Aβ1–42): 4G8 reacted with peptides corresponding to residues 17–21, 6F/3D reacted with peptides corresponding to residues 9–14, and anti 5-10 reacted with peptides corresponding to residues 5–10. The study also yielded some insight into the Aβ1–42 structures resulting from differences in pH. An ELISA study using monoclonal antibodies showed that pH-dependent conformational changes occur in the 6F/3D and 4G8 epitopes modified at pH 4.6, but not in the sequences recognized by anti 1-7 and anti 5-10. This was unique to Aβ1–40 and Aβ1–42 and did not occur with Aβ1–16 or Aβ17–42. The reactivity profile of 4G8 was not affected by blockage of histidine residues of pH-modified Aβ1–40 and Aβ1–42 with diethyl pyrocarbonate; however, the mutant [Gln11]Aβ1–40 abrogated the unique pH-dependence towards 4G8 observed with Aβ1–40. These findings suggest that these epitopes are cryptic at pH4.6, and that Glu11 is responsible for the changes. We suggest that the abnormal folding of 6F/3D epitope affected by pH masked the 4G8 epitope. A study of the binding of metal ions to Aβ1–42 suggested that Cu2+ and Zn2+ induced a conformational transition around the 6F/3D region at pH7.4, but did not affect the region when it was modified at pH4.6. However, Fe2+ had no effect, irrespective of pH. Aβ modified at pH 4.6 appeared to be relatively resistant to proteinase K compared with Aβs modified at pH7.4, and the former might be preferentially internalized and accumulated in a human glial cell. Our findings suggest the importance of microenvironmental changes, such as pH, in the early stage of formation of Aβ aggregates in the glial cell.

scholarly journals Tertiary Conformational Transition In Horse Haemoglobin Induced By Inositol Hexakisphosphate

2013 ◽  
Vol 27 (S1) ◽  
Author(s):  
Omolola E Omotosho ◽  
Kehinde O. Okonjo ◽  
Victor T. Omotosho ◽  
Solomon O. Rotimi ◽  
Shalom N. Chinedu
Keyword(s):  
Conformational Transition ◽  
Inositol Hexakisphosphate

scholarly journals Conformational Transition of KcsA Gating and the Mechanism of its pH-Dependence

2013 ◽  
Vol 104 (2) ◽  
pp. 25a
Author(s):  
Po-Chao Wen ◽  
Mahmoud Moradi ◽  
Raymond E. Hulse ◽  
Eduardo Perozo ◽  
Emad Tajkhorshid
Keyword(s):  
Conformational Transition ◽  
Ph Dependence

scholarly journals The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre

1999 ◽  
Vol 338 (1) ◽  
pp. 235-239 ◽  
Author(s):  
Manuel MORILLAS ◽  
Martin L. GOBLE ◽  
Richard VIRDEN
Keyword(s):  
Rate Constant ◽  
Conformational Transition ◽  
Ph Dependence ◽  
Penicillin Acylase ◽  
Penicillin G ◽  
Pka Values ◽  
Energy Compensation ◽  
Guanidinium Group ◽  
Kinetics Of ◽  
Hydrolysis Of

Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a kcat of 0.8 s-1 and a Km of 10 µM at pH 7.5 and 20 °C. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s-1 and a subsequent deacylation step with a rate constant of 0.81 s-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to KS was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 °C, the pH-dependence of kcat was similar to that of kcat/Km, indicating that formation of the acyl-enzyme did not affect the pKa values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pKa for kcat, which measures the rate of deacylation, are consistent with α-amino and guanidinium groups whose pKa values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the α-amino group of the catalytic SerB1 and the guanidinium group of ArgB263 are required in neutral and protonated states respectively.

Sign in / Sign up

Export Citation Format

Share Document