Biochemistry & Molecular Biology

Towards the Preparation of a Cysteine-free Creatine Kinase Suitable for FRET Studies

Document Type

Poster Presentation

Location

Indianapolis, IN

Start Date

13-4-2018 2:30 PM

End Date

13-4-2018 4:00 PM

Description

Creatine kinase (CK), found in all vertebrates, catalyzes the reversible interconversion of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine can be viewed as a reservoir of “high energy phosphate”, able provide ATP on demand. There are two soluble isozymes of creatine kinase, muscle and brain, both of which form homodimers, MMCK and BBCK, respectively. While the enzyme is well characterized, X-ray structures of MM creatine kinase from the electric ray, Torpedo californica, show significant conformational changes during catalysis. Specifically, there are two loops that move considerable to covering the active site during phosphate transfer. This movement is of additional interest because residues that are quite distant in the substrate-free enzyme also appear to play a role in in substrate recognition. It is thought that fluorescence resonance energy transfer (FRET) may prove useful in tracking movements of these loops. This technique is made possible by the use of fluorescent dyes attached to the enzyme in specific positions through reaction with cysteine residues. To ensure correct labeling, cysteine residues from the native enzyme must be removed. Preliminary studies indicated that complete replacement of all non-active site cysteines with serine resulted in insoluble protein. In this project, we have focused on addressing the issues caused by the insolubility. This includes the use of histidine and GST tags to improve the purification methodology for both the wild-type CK and its variants. Biochemistry & Molecular Biology

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Apr 13th, 2:30 PM Apr 13th, 4:00 PM

Towards the Preparation of a Cysteine-free Creatine Kinase Suitable for FRET Studies

Indianapolis, IN

Creatine kinase (CK), found in all vertebrates, catalyzes the reversible interconversion of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine can be viewed as a reservoir of “high energy phosphate”, able provide ATP on demand. There are two soluble isozymes of creatine kinase, muscle and brain, both of which form homodimers, MMCK and BBCK, respectively. While the enzyme is well characterized, X-ray structures of MM creatine kinase from the electric ray, Torpedo californica, show significant conformational changes during catalysis. Specifically, there are two loops that move considerable to covering the active site during phosphate transfer. This movement is of additional interest because residues that are quite distant in the substrate-free enzyme also appear to play a role in in substrate recognition. It is thought that fluorescence resonance energy transfer (FRET) may prove useful in tracking movements of these loops. This technique is made possible by the use of fluorescent dyes attached to the enzyme in specific positions through reaction with cysteine residues. To ensure correct labeling, cysteine residues from the native enzyme must be removed. Preliminary studies indicated that complete replacement of all non-active site cysteines with serine resulted in insoluble protein. In this project, we have focused on addressing the issues caused by the insolubility. This includes the use of histidine and GST tags to improve the purification methodology for both the wild-type CK and its variants. Biochemistry & Molecular Biology