U i Using mutagenesis to assist in NMR t i t i t i NMR assignment of - PowerPoint PPT Presentation

U i Using mutagenesis to assist in NMR t i t i t i NMR assignment of chemotaxis receptor Sergey Shnitkind Los Angeles Valley College Biochemistry Mentor Anh Vu Mentor: Anh Vu Dr. Dahlquist’s Lab Chemistry Department Chemistry Department

B Background information k d i f ti Project goal j g Methods Results

What is chemotaxis? Movement of bacteria towards or away Movement of bacteria towards or away from chemical stimuli. Gather knowledge E Coli Flagella E. Coli Flagella Receptors Applications Inside cytoplasm Prevent/inhibit diseases Chemotaxis receptor; TM14 Brian R. Crane et. al.

To create a single mutation in the chemotaxis To create a single mutation in the chemotaxis protein receptor to assist in methyl side chain assignment of the receptor protein assignment of the receptor protein. C13 Chemical Shift (ppm) H1 Chemical Shift (ppm) Sample 2D NMR spectrum Chemotaxis receptor TM14; http://www.pdb.org

To create a single mutation in the chemotaxis To create a single mutation in the chemotaxis protein receptor to assist in methyl side chain assignment of the receptor protein assignment of the receptor protein. C13 Chemical Shift (ppm) H1 Chemical Shift (ppm) Sample 2D NMR spectrum Chemotaxis receptor TM14; http://www.pdb.org

Various pictures from our lab at UCSB

Create mutant receptors using PCR  C  Custom primers t i  Single nucleotide mutation  Single nucleotide mutation PCR in our lab  Single amino ‐ acid mutation g  Ex: TTA to ATA (Leucine to Isoleucine) PCR diagram: http://www.obgynacademy.com/

Protein expression  Expression = Production of desired protein P d ti f d i d t i by the bacteria. Protein purification  Using nickel column to bind protein.  Elute protein from column using another chemical that competes for nickel binding for nickel binding. Nickel column in our lab

Obtain NMR spectrum  D 1 H  2D 1 H – 13 C NMR 13 C NMR  Each nucleus will give a  Each nucleus will give a different signal based on its environment Our NMR Machine at UCSB  Comparing spectrum from wildtype and mutant will allow us to verify correct assignment of peaks ll if i f k

Various pictures from our lab at UCSB

Successfully created mutant receptors Successfully created mutant receptors  Single amino ‐ acid mutation  Results verified using  Results verified using DNA sequencing. DNA Plasmid ~5kbp ..GEAGKGFMLVANEVQNLSNETN.. ..GEAGKGFMIVANEVQNLSNETN.. ..GEAGKGFMIVANEVQNLSNETN.. ..******** *************.. Sin Single amino-acid mutation g le amino-acid mutation Agarose gel 1%, purified DNA plasmid. Taken at our lab.

Successfully expressed mutant receptors Successfully expressed mutant receptors  Our receptor is clearly expressed  Our receptor is clearly expressed (compared to no expression). kDa 170 70 40 35 25 15 Receptor ~13kDa ~13kDa 10 No expression SDS ‐ PAGE of expressed receptors; SDS ‐ PAGE of unexpressed receptors. Taken at our lab.

Mutant Wildtype Wildtype NMR spectrum of a mutant receptor, taken in our lab at UCSB by Anh Vu.

Amino acid #156 Amino acid #156 Isoleucine (wildtype) Mutant Amino acid #156 Wildtype Wildtype Leucine (mutant) NMR spectrum of a mutant receptor, taken in our lab at UCSB by Anh Vu.

Amino acid #156 Isoleucine (wildtype) Amino acid #156 Leucine (mutant) Mutant Wildtype NMR spectrum of a mutant receptor, taken in our lab at UCSB by Anh Vu.

Dahlquist’s Lab Mentor: Anh Vu Lab Partner: Melanie Shelton b l h l (NMR spectrum is of Melanie’s mutant) Dr Frederick Dahlquist Dr. Frederick Dahlquist INSET Program Dr Jens ‐ Uwe Kuhn Dr. Jens ‐ Uwe Kuhn Dr. Nicholas Arnold Dr Arica Lubin Dr. Arica Lubin

I f Infected species t d i Ch Chmotactic t ti S Symptoms/ t / Bacteria Diseases Various crops p Dickeya dadantii y Necrosis (potato, chicory) Mammals (inc. humans) Spirochetes Lyme disease, relapsing fever, l f syphilis Fish, humans Fish humans Vibrio Vibrio Various infections Various infections Humans Helicobacter Inflammation, pylori Ulcers

001 MGSYHHHHHHSSGLVPRGSHMKSGTNVDQIVERVKEASSQIGETLENIRSIEKLIQNIMR 001 MGSYHHHHHHSSGLVPRGSHMKSGTNVDQIVERVKEASSQIGETLENIRSIEKLIQNIMR 001 ************************************************************ 061 IARETNILALNATIEAARAGEAGKGFMLVANEVQNLSNETNEVTKQIVEKAREILESSQR 061 IARETNILALNATIEAARAGEAGKGFMIVANEVQNLSNETNEVTKQIVEKAREILESSQR 061 *************************** ******************************** 121 SLENLEFMANLFETVGKT 121 SLENLEFMANLFETVGKT 121 ****************** Top- Top- Mutant Mutant Bottom Bottom Bottom Bottom- Wildtype Wildtype Wildtype Wildtype Single amino-acid mutation. Single amino-acid mutation.

Mutant Wildtype Wildtype NMR spectrum of a mutant receptor, taken in our lab at UCSB.