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SRX19576618: GSM7082772: nonselect_library_replicate2_tile11 (81); synthetic construct; OTHER
1 ILLUMINA (NextSeq 550) run: 3.8M spots, 1.1G bases, 297.8Mb downloads

External Id: GSM7082772_r1
Submitted by: University of Copenhagen
Study: Characterizing glucokinase variant mechanisms using a multiplexed abundance assay
show Abstracthide Abstract
Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms of human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity. We assayed the abundance of 95\% GCK missense and nonsense variants, and found that 59\% of hypoactive variants have a decreased cellular abundance. By combining our abundance scores with predictions of protein thermodynamic stability, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis. Overall design: Three regional libraries of human glucokinase variants (aa 2-171, 172-337, 338-465) were transformed into yeast (BY4741), and were grown on SC+leucine+methionine+histidine medium with methotrexate to select for glucokinase variant abundance. The variants were sequenced before and after selection using 14 tiles spanning the glucokinase ORF to determine their change in frequency and thereby effect on protein abundance.
Sample: nonselect_library_replicate2_tile11 (81)
SAMN33602943 • SRS16959802 • All experiments • All runs
Library:
Name: GSM7082772
Instrument: NextSeq 550
Strategy: OTHER
Source: OTHER
Selection: other
Layout: PAIRED
Construction protocol: For all conditions, plasmid DNA was extracted from 9 OD units of yeast cells using the ChargeSwitch Plasmid Yeast Mini kit (Invitrogen). The Gateway entry libraries were previously constructed (Gersing et al., 2022, bioRxiv) and were cloned into the pDEST-DHFR-PCA Gateway destination vector.
Runs: 1 run, 3.8M spots, 1.1G bases, 297.8Mb
Run# of Spots# of BasesSizePublished
SRR237153943,782,1621.1G297.8Mb2023-05-22

ID:
26869239

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