Progress on the co-crystallization of Thermoplasma acidophilum - - PowerPoint PPT Presentation

▶
progress on the co crystallization
SMART_READER_LITE
LIVE PREVIEW

Progress on the co-crystallization of Thermoplasma acidophilum - - PowerPoint PPT Presentation

Mar 24, 2023 50 likes •171 views

Progress on the co-crystallization of Thermoplasma acidophilum nucleoside kinase (TaNK) with substrates Jessica Yoo Columbus Lab CSS Symposium 1 Protein Misannotation High as for some protein families 1 80% Accuracy

slide-1
SLIDE 1

Progress on the co-crystallization

  • f Thermoplasma acidophilum

nucleoside kinase (TaNK) with substrates

Jessica Yoo Columbus Lab CSS Symposium

1

slide-2
SLIDE 2

Protein Misannotation

  • High as for some protein families1
  • Accuracy
  • Time, man-hours, money

80%

2

slide-3
SLIDE 3

Why TaNK?

  • 1. Nucleoside kinase vs. ribokinase
  • 2. Broad-range specificity

Improve our understanding of structure-function relationships

3

slide-4
SLIDE 4

Production of Protein

4

slide-5
SLIDE 5

Immobilized Metal Affinity Chromatography gel confirmation

Adapted from bioenergy.asu.edu 5 34.94 kDa

slide-6
SLIDE 6

chromatogram

Size Exclusion Chromatography

Adapted from en.wikipedia.org 6

slide-7
SLIDE 7

Chromatogram

7

slide-8
SLIDE 8

Crystallization

Adapted from www.bio.davidson.edu

“drying agent”

8

slide-9
SLIDE 9

1 nanoliter 9

slide-10
SLIDE 10

Present and Future

  • Protein successfully expressed and purified
  • Protein crystallized in nL quantities
  • Crystallization conditions optimized
  • nL  μL
  • More screening for optimal conditions
  • X-ray crystallography

10

slide-11
SLIDE 11

Acknowledgements

  • Professor Linda Columbus & the Columbus-Mura Lab
  • Charles Henry Leach, II Foundation; Wendy R. Van

Besien and Stephen M. Van Besien; Lois A. Fitton & Christopher Draper, Jr.; Sharon B. Parente & John W. Risner; The Jefferson Trust; Mary Baroody Lowe and Jeffrey A. Lowe; J. Randolph and Rossie Carter Hutcheson; Entigence Corporation; Robert Atkinson

  • The UVa Parents Fund and Committee
  • The College Science Scholars Program

11

slide-12
SLIDE 12

Bibliography

1. Schnoes, A. M., Brown, S. D., Dodevski, I., & Babbitt, P. C. (2009). Annotation Error in Public Databases: Misannotation of Molecular Function in Enzyme Superfamilies. PLoS Comput Biol, 5(12),

  • e1000605. doi:10.1371/journal.pcbi.1000605

2. Elkin, S. R., Kumar, A., Price, C. W., & Columbus, L. (2013). A broad specificity nucleoside kinase from Thermoplasma

  • acidophilum. Proteins, 81(4), 568–582. doi:10.1002/prot.24212

3. Ota H, Sakasegawa S, Yasuda Y, Imamura S, Tamura T. A novel nucleo- side kinase from Burkholderia thailandensis: a member of the phospho- fructokinase B-type family of enzymes. FEBS J 2008;275: 5865–5872. 4. Hansen T, Arnfors L, Ladenstein R, Schonheit P. The phosphofruc- tokinase-B (MJ0406) from Methanocaldococcus jannaschii represents a nucleoside kinase with a broad substrate specificity. Extremophiles 2007;11:105–114.

12