Biology subjects

Borkowska, O.

Publications and source records attributed to Borkowska, O..

3 recordsLinked to original sources

bioRxiv2019

Electrospray Surface Charge Describes Protein Molecular Motion

The electrospray mass/charge distribution for a protein is an instantaneous measurement of surface areas during the transition from liquid to gas phase. Protonation is dependent upon surface area and surface area is related to protein folding under all conditions. M/z distributions for proteins and protein analogues all with different degrees of entropy were compared. Rigid dipeptide self-assemblies of any size possess a single m/z, whereas protein always displays multiple m/z distributions. Native proteins have narrow, defined m/z distributions, while denatured proteins and synthetic homopolypetides possess the widest possible m/z distributions. These observations are consistent with dynamic changes in surface area resulting from molecular motion.

biophysics↗

bioRxiv2018

Structural basis of human 5,10-methylenetetrahydrofolate reductase (MTHFR) regulation by phosphorylation and S-adenosylmethionine inhibition

The folate and methionine cycles are crucial to the biosynthesis of lipids, nucleotides and proteins, and production of the global methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 [A] resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region into proximity with the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16-total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation.

biochemistry↗

bioRxiv2018

Zinc(II) binding on human wild-type ISCU and Met140 variants modulates Fe-S complex activity

The human de novo iron-sulfur (Fe-S) assembly complex consists of the cysteine desulfurase NFS1, accessory protein ISD11, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN has been shown to bind the NFS1-ISD11-ISCU complex (SDU), to activate the desulfurase activity and thus Fe-S cluster biosynthesis. Conversely, in the absence of FXN, the NFS1-ISD11 (SD) complex was reported to be inhibited by the binding of recombinant ISCU. Here, we show that recombinant ISCU binds zinc(II) ion, and that the presence of zinc in as-isolated ISCU has impacts on the SDU desulfurase activity as measured by sulfide production. Indeed, the removal of this zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SD alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SD. Recent yeast studies have reported a substitution on the yeast ISCU orthologue Isu, at position Met141 (Met140 in human numbering of precursor protein) to Ile, Leu, Val, or Cys that could bypass the requirement of FXN for Fe-S cluster assembly and cell viability. Using recombinant human proteins, we report no significant differences in the biochemical and biophysical properties observed between wild-type and variants M140I, M140 L, and M140 V of ISCU. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SD complex. This study therefore identifies an important regulatory role for ISCU-bound zinc in modulation of the human Fe-S assembly system in vitro but no FXN bypass effect on mutations at position Met140 in human ISCU.\n\nABBREVIATIONS

biochemistry↗