scholarly journals A topologically conserved aliphatic residue in α-helix 6 stabilizes the hydrophobic core in domain II of glutathione transferases and is a structural determinant for the unfolding pathway

1998 ◽  
Vol 336 (2) ◽  
pp. 413-418 ◽  
Author(s):  
Louise A. WALLACE ◽  
Gregory L. BLATCH ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Fluorescence Studies ◽  
Subunit Dissociation ◽  
Equilibrium Unfolding ◽  
Α Helix ◽  
Dimeric Intermediate ◽  
Conserved Residue ◽  
Structural Determinant

A topologically conserved residue in α-helix 6 of domain II of human glutathione transferase (hGST) A1-1 was mutated to investigate its contribution to protein stability and the unfolding pathway. The replacement of Leu-164 with alanine (L164A) did not impact on the functional and gross structural properties of native hGST A1-1. The wild-type protein unfolds via a three-state pathway in which only folded dimer and unfolded monomer were highly populated at equilibrium; a native-like dimeric intermediate with partially dissociated domains I and II was detected using stopped-flow fluorescence studies [Wallace, Sluis-Cremer and Dirr (1998) Biochemistry 37, 5320–5328]. In the present study, urea-induced equilibrium unfolding of L164A hGST A1-1 indicated a destabilization of the native state and suggested the presence of a stable dimeric intermediate. The unfolding kinetic pathway for L164A hGST A1-1, like that for the wild type, is biphasic, with a fast and a slow unfolding event; the cavity-forming mutation has a substantially greater effect on the rate of unfolding of the fast event. The equilibrium and kinetic unfolding data for L164A hGST A1-1 suggest that a rapid pre-equilibrium is established between the native dimer and a dimeric intermediate before complete domain and subunit dissociation and unfolding. It is proposed that the topologically conserved bulky residue in α-helix 6 plays a role in specifying and stabilizing the core of domain II and the interface of domains I and II.

scholarly journals The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

2003 ◽  
Vol 372 (1) ◽  
pp. 241-246 ◽  
Author(s):  
Chris NATHANIEL ◽  
Louise A. WALLACE ◽  
Jonathan BURKE ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Conformational Stability ◽  
Wild Type ◽  
Catalytic Function ◽  
Evolutionarily Conserved ◽  
Glutathione S Transferases ◽  
Equilibrium Unfolding ◽  
The Stability ◽  
Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Steven W. Pipe ◽  
Evgueni L. Saenko ◽  
Angela N. Eickhorst ◽  
Geoffrey Kemball-Cook ◽  
Randal J. Kaufman
Keyword(s):  
Protein Interactions ◽  
Hemophilia A ◽  
Specific Activity ◽  
Homology Model ◽  
Optical Biosensor ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Activity Assay ◽  
Subunit Dissociation ◽  
A Domains

Abstract Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino acid residues 373-740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, ARG531HIS, is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted ARG531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting ARG531 (ALA284GLU, ALA284PRO) or closely adjacent to the A1-A2 interface in the tightly packed hydrophobic core (SER289LEU) have the same phenotype of 1-stage/2-stage discrepancy. TheALA284GLU andSER289LEU mutations in FVIII were produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type both mutants had reduced specific activity by 1-stage clotting activity and at least a 2-fold lower activity by 2-stage analysis (COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified ALA284GLU andSER289LEU proteins in an optical biosensor demonstrated that A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the ARG531HISmutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa.

Lipid interactions with bacterial channels: fluorescence studies

2005 ◽  
Vol 33 (5) ◽  
pp. 905-909 ◽  
Author(s):  
A.M. Powl ◽  
J. Carney ◽  
P. Marius ◽  
J.M. East ◽  
A.G. Lee
Keyword(s):  
Fluorescence Quenching ◽  
Hot Spot ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Hydrophobic Core ◽  
Fluorescence Studies ◽  
Potassium Channel Kcsa ◽  
Α Helix

Interactions between a membrane protein and the lipid molecules that surround it in the membrane are important in determining the structure and function of the protein. These interactions can be pictured at the molecular level using fluorescence spectroscopy, making use of the ability to introduce tryptophan residues into regions of interest in bacterial membrane proteins. Fluorescence quenching methods have been developed to study lipid binding separately on the two sides of the membrane. Lipid binding to the surface of the mechanosensitive channel MscL is heterogeneous, with a hot-spot for binding anionic lipid on the cytoplasmic side, associated with a cluster of three positively charged residues. The environmental sensitivity of tryptophan fluorescence emission has been used to identify the residues at the ends of the hydrophobic core of the second transmembrane α-helix in MscL. The efficiency of hydrophobic matching between MscL and the surrounding lipid bilayer is high. Fluorescence quenching methods can also be used to study binding of lipids to non-annular sites such as those between monomers in the homotetrameric potassium channel KcsA.

scholarly journals Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deficient Mutant Acclimated to Low and High Temperatures

Biomolecules ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 27
Author(s):  
Iwona Sadura ◽  
Dariusz Latowski ◽  
Jana Oklestkova ◽  
Damian Gruszka ◽  
Marek Chyc ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Temperature Stress ◽  
High Temperatures ◽  
Biological Membrane ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Chloroplast Membranes ◽  
Paramagnetic Resonance ◽  
Photosynthetic Membranes

Plants have developed various acclimation strategies in order to counteract the negative effects of abiotic stresses (including temperature stress), and biological membranes are important elements in these strategies. Brassinosteroids (BR) are plant steroid hormones that regulate plant growth and development and modulate their reaction against many environmental stresses including temperature stress, but their role in modifying the properties of the biological membrane is poorly known. In this paper, we characterise the molecular dynamics of chloroplast membranes that had been isolated from wild-type and a BR-deficient barley mutant that had been acclimated to low and high temperatures in order to enrich the knowledge about the role of BR as regulators of the dynamics of the photosynthetic membranes. The molecular dynamics of the membranes was investigated using electron paramagnetic resonance (EPR) spectroscopy in both a hydrophilic and hydrophobic area of the membranes. The content of BR was determined, and other important membrane components that affect their molecular dynamics such as chlorophylls, carotenoids and fatty acids in these membranes were also determined. The chloroplast membranes of the BR-mutant had a higher degree of rigidification than the membranes of the wild type. In the hydrophilic area, the most visible differences were observed in plants that had been grown at 20 °C, whereas in the hydrophobic core, they were visible at both 20 and 5 °C. There were no differences in the molecular dynamics of the studied membranes in the chloroplast membranes that had been isolated from plants that had been grown at 27 °C. The role of BR in regulating the molecular dynamics of the photosynthetic membranes will be discussed against the background of an analysis of the photosynthetic pigments and fatty acid composition in the chloroplasts.

scholarly journals Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners

2021 ◽  
Vol 22 (19) ◽  
pp. 10805
Author(s):  
José L. Neira ◽  
David Ortega-Alarcón ◽  
Bruno Rizzuti ◽  
Martina Palomino-Schätzlein ◽  
Adrián Velázquez-Campoy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Antibacterial Properties ◽  
Helical Structure ◽  
Titration Calorimetry ◽  
Its Sequence ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Residual Structure ◽  
Α Helix

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect

2002 ◽  
Vol 362 (3) ◽  
pp. 749-754 ◽  
Author(s):  
Ziedulla Kh. ABDULLAEV ◽  
Marina E. BODROVA ◽  
Boris V. CHERNYAK ◽  
Dmitry A. DOLGIKH ◽  
Ruth M. KLUCK ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Antioxidant Activities ◽  
H2o2 Production ◽  
High Electron ◽  
Wild Type ◽  
Low Concentrations ◽  
Rat Heart Mitochondria ◽  
The Difference ◽  
Α Helix

A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal α-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H2O2 production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other ‘horse’ modifications in the N-terminal α-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2–12.

scholarly journals Three Residues in HIV-1 Matrix Contribute to Protease Inhibitor Susceptibility and Replication Capacity

2010 ◽  
Vol 55 (3) ◽  
pp. 1106-1113 ◽  
Author(s):  
Chris M. Parry ◽  
Madhavi Kolli ◽  
Richard E. Myers ◽  
Patricia A. Cane ◽  
Celia Schiffer ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cleavage Site ◽  
Matrix Protein ◽  
Resistant Mutant ◽  
Drug Susceptibility ◽  
Hiv Protease ◽  
Wild Type Virus ◽  
Replication Capacity ◽  
Wild Type ◽  
Α Helix

ABSTRACTOther than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations ingag, particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5- to 7-fold change in protease inhibitor 50% effective concentration (EC50). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within α-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.

scholarly journals Palmitoylation of Apolipoprotein B Is Required for Proper Intracellular Sorting and Transport of Cholesteroyl Esters and Triglycerides

2000 ◽  
Vol 11 (2) ◽  
pp. 721-734 ◽  
Author(s):  
Yang Zhao ◽  
James B. McCabe ◽  
Jean Vance ◽  
Luc G. Berthiaume
Keyword(s):  
Apolipoprotein B ◽  
Neutral Lipids ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Very Low Density Lipoproteins ◽  
Structural Requirement ◽  
Lipoprotein Particles ◽  
Intracellular Sorting

Apolipoprotein B (apoB) is an essential component of chylomicrons, very low density lipoproteins, and low density lipoproteins. ApoB is a palmitoylated protein. To investigate the role of palmitoylation in lipoprotein function, a palmitoylation site was mapped to Cys-1085 and removed by mutagenesis. Secreted lipoprotein particles formed by nonpalmitoylated apoB were smaller and denser and failed to assemble a proper hydrophobic core. Indeed, the relative concentrations of nonpolar lipids were three to four times lower in lipoprotein particles containing mutant apoB compared with those containing wild-type apoB, whereas levels of polar lipids isolated from wild-type or mutant apoB lipoprotein particles appeared identical. Palmitoylation localized apoB to large vesicular structures corresponding to a subcompartment of the endoplasmic reticulum, where addition of neutral lipids was postulated to occur. In contrast, nonpalmitoylated apoB was concentrated in a dense perinuclear area corresponding to the Golgi compartment. The involvement of palmitoylation as a structural requirement for proper assembly of the hydrophobic core of the lipoprotein particle and its intracellular sorting represent novel roles for this posttranslational modification.

Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors

1987 ◽  
Vol 53 (1-2) ◽  
pp. 33-44 ◽  
Author(s):  
U. Gehring ◽  
K. Mugele ◽  
H. Arndt ◽  
W. Busch
Keyword(s):  
Glucocorticoid Receptors ◽  
Wild Type ◽  
Subunit Dissociation
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