Influence of solution ionic strength on the stabilities of M20 loop conformations in apo E. coli dihydrofolate reductase

2021 ◽  
Vol 154 (19) ◽  
pp. 195103
Author(s):  
C. Satheesan Babu ◽  
Carmay Lim
Keyword(s):  
Ionic Strength ◽  
Dihydrofolate Reductase ◽  
Solution Ionic Strength ◽  
E Coli ◽  
Apo E ◽  
Loop Conformations

High-resolution gold labeling

1993 ◽  
Vol 51 ◽  
pp. 330-331
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya ◽  
Kyra Carbone ◽  
Martha Simon ◽  
Beth Lin ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Polypeptide Chain ◽  
External Surface ◽  
Gold Cluster ◽  
Macromolecular Complex ◽  
Gold Compound ◽  
Central Cavity ◽  
Chain Folding ◽  
E Coli ◽  
Chaperonin Groel

A recently developed 1.4 nm gold cluster has been found to be useful in labeling macromolecular sites to 1-3 nm resolution. The gold compound is organically derivatized to contain a monofunctional arm for covalent linking to biomolecules. This may be used to mark a specific site on a structure, or to first label a component and then reassemble a multicomponent macromolecular complex. Two examples are given here: the chaperonin groEL and ribosomes.Chaperonins are essential oligomeric complexes that mediate nascent polypeptide chain folding to produce active proteins. The E. coli chaperonin, groEL, has two stacked rings with a central hole ∽6 nm in diameter. The protein dihydrofolate reductase (DHFR) is a small protein that has been used in chain folding experiments, and serves as a model substrate for groEL. By labeling the DHFR with gold, its position with respect to the groEL complex can be followed. In particular, it was sought to determine if DHFR refolds on the external surface of the groEL complex, or whether it interacts in the central cavity.

Temperature-Regulated Fluorescence and Association of an Oligo(ethyleneglycol)methacrylate-Based Copolymer with a Conjugated Polyelectrolyte—The Effect of Solution Ionic Strength

2013 ◽  
Vol 117 (46) ◽  
pp. 14576-14587 ◽  
Author(s):  
Sahika Inal ◽  
Leonardo Chiappisi ◽  
Jonas D. Kölsch ◽  
Mario Kraft ◽  
Marie-Sousai Appavou ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Conjugated Polyelectrolyte ◽  
Solution Ionic Strength

A structure—function study of dihydrofolate reductase by protein engineering

1986 ◽  
Vol 317 (1540) ◽  
pp. 405-413 ◽  
Keyword(s):  
Structure Function ◽  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Guanidine Hydrochloride ◽  
Disulphide Bond ◽  
E Coli ◽  
Transition State Stabilization ◽  
Disulphide Bond Formation ◽  
High Resolution Structure ◽  
Charge Interaction

Several mutants of the enzyme dihydrofolate reductase (DHFR) have been engineered by oligonucleotide-directed mutagenesis of the cloned E. coli gene. The mutations were designed to address specific questions about DHFR structure-function relations that arose from the analysis of the high-resolution structure. Mutations at the active site have revealed that the invariant residue aspartate-27 is involved in substrate protonation, and not in transition-state stabilization as previously thought. The 2.0 Å (1 Å = 10 -1 nm = 10 -10 m) refined structures of the Asn-27 and Ser-27 mutant enzymes reveal that the enhanced binding observed for the 2,4-diamino pteridine and pyrimidine inhibitors is probably not attributable to the charge interaction between Asp-27 and a protonated N-1 of the inhibitor. Substitution of a cysteine for a proline at position 39 places two sulphydryls within bonding distance, and under certain oxidation conditions they will quantitatively form a disulphide bond. The refined 2.0 Å structures of both reduced and oxidized forms of this mutant show that only minor conformational changes occur for disulphide bond formation. The crosslinked enzyme is significantly more conformationally stable to denaturants such as guanidine hydrochloride and urea.

scholarly journals Switching an active site helix in dihydrofolate reductase reveals limits to sub-domain modularity

2021 ◽  
Author(s):  
Victor Y. Zhao ◽  
João V. Rodrigues ◽  
Elena R. Lozovsky ◽  
Daniel L. Hartl ◽  
Eugene I. Shakhnovich
Keyword(s):  
Catalytic Activity ◽  
Dihydrofolate Reductase ◽  
Strongly Correlated ◽  
Thermal Stabilities ◽  
E Coli ◽  
In Vitro Measurements ◽  
Flux Model ◽  
Α Helix ◽  
Dynamics Simulations

To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study sub-domain modularity by creating 20 chimeras of an enzyme, E. coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR. We performed molecular dynamics simulations using replica exchange with solute-tempering. Chimeras with low catalytic activity exhibit non-helical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.

Ionic strength and polyelectrolyte molecular weight effects on floc formation and growth in Taylor–Couette flows

Soft Matter ◽  
2021 ◽  
Author(s):  
Athena E. Metaxas ◽  
Vishal Panwar ◽  
Ruth L. Olson ◽  
Cari S. Dutcher
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Solution Ionic Strength ◽  
Radial Injection ◽  
Couette Cell ◽  
Taylor Couette

A Taylor–Couette cell capable of radial injection was used to study the effects of varying solution ionic strength and polyelectrolyte molecular weight on the polyelectrolyte-driven flocculation of bentonite suspensions.

scholarly journals Studies on the Interaction between Poly-Phosphane Gold(I) Complexes and Dihydrofolate Reductase: An Interplay with Nicotinamide Adenine Dinucleotide Cofactor

2019 ◽  
Vol 20 (7) ◽  
pp. 1802
Author(s):  
Stefania Pucciarelli ◽  
Silvia Vincenzetti ◽  
Massimo Ricciutelli ◽  
Oumarou Camille Simon ◽  
Anna Teresa Ramadori ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interactions ◽  
Dissociation Constants ◽  
Gold Compound ◽  
Inhibitory Effects ◽  
Binding Properties ◽  
Enzyme Active Site ◽  
E Coli ◽  
Kd Values ◽  
Gold Compounds

A class of gold(I) phosphane complexes have been identified as inhibitors of dihydrofolate reductase (DHFR) from E. coli, an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. In this work, to comprehend the nature of the interaction at the basis of these inhibitory effects, the binding properties of bis- and tris-phosphane gold(I) chloride compounds in regards to DHFR have been studied by emission spectroscopy and spectrophotometric assays. The lack of cysteine and seleno-cysteine residues in the enzyme active site, the most favorable sites of attack of Au(I) moieties, makes this work noteworthy. The interaction with the gold compounds results into the quenching of the DHFR tryptophan’s emissions and in an enhancement of their intrinsic emission intensities. Moreover, a modulating action of NADPH is highlighted by means of an increase of the gold compound affinity toward the enzyme; in fact, the dissociation constants calculated for the interactions between DHFR and each gold compound in the presence of saturating NADPH were lower than the ones observed for the apo-enzyme. The fluorimetric data afforded to Kd values ranged from 2.22 ± 0.25 µM for (PPh3)2AuCl in the presence of NADPH to 21.4 ± 3.85 µM for 4L3AuTf in the absence of NADPH. By elucidating the energetic aspects of the binding events, we have attempted to dissect the role played by the gold phosphane/protein interactions in the inhibitory activity, resulting in an exothermic enthalpy change and a positive entropic contribution (ΔH° = −5.04 ± 0.08 kcal/mol and ΔS° = 7.34 ± 0.005 cal/mol·K).

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