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U i t i t i t i NMR Using mutagenesis to assist in NMR assignment of chemotaxis receptor Sergey Shnitkind Los Angeles Valley College Biochemistry Mentor Anh Vu Mentor: Anh Vu
- Dr. Dahlquist’s Lab
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 - - 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. Dahlquists Lab Chemistry Department Chemistry Department B
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What is chemotaxis?
Movement of bacteria towards or away Movement of bacteria towards or away from chemical stimuli.
Gather knowledge
E Coli Flagella
Applications
- E. Coli Flagella
Receptors Inside cytoplasm
Prevent/inhibit diseases
Chemotaxis receptor; TM14 Brian R. Crane et. al.
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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)
Chemotaxis receptor TM14; http://www.pdb.org Sample 2D NMR spectrum
H1 Chemical Shift (ppm)
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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)
Chemotaxis receptor TM14; http://www.pdb.org Sample 2D NMR spectrum
H1 Chemical Shift (ppm)
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Various pictures from our lab at UCSB
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Create mutant receptors using PCR
C
t i
Custom primers Single nucleotide mutation Single nucleotide mutation Single amino‐acid mutation
PCR in our lab
g
Ex: TTA to ATA (Leucine to Isoleucine)
PCR diagram: http://www.obgynacademy.com/
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Protein expression
Expression =
P d ti f d i d t i Production of desired protein 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
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Obtain NMR spectrum
D 1H
13C NMR
2D 1H – 13C NMR Each nucleus will give a Each nucleus will give a
different signal based on its environment
Comparing spectrum from wildtype and mutant will
ll if i f k
Our NMR Machine at UCSB
allow us to verify correct assignment of peaks
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Various pictures from our lab at UCSB
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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 le amino-acid mutation
Agarose gel 1%, purified DNA plasmid. Taken at our lab.
g
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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 SDS‐PAGE of expressed receptors; SDS‐PAGE of unexpressed receptors. Taken at our lab. 10 ~13kDa No expression
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Mutant Wildtype
NMR spectrum of a mutant receptor, taken in our lab at UCSB by Anh Vu.
Wildtype
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Amino acid #156 Amino acid #156 Isoleucine (wildtype)
Mutant Wildtype
Amino acid #156 NMR spectrum of a mutant receptor, taken in our lab at UCSB by Anh Vu.
Wildtype
Leucine (mutant)
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Amino acid #156 Isoleucine (wildtype) Amino acid #156 Leucine (mutant) NMR spectrum of a mutant receptor, taken in our lab at UCSB by Anh Vu. Mutant Wildtype
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Dahlquist’s Lab Mentor: Anh Vu b l h l Lab Partner: Melanie Shelton
(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
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I f t d i Ch t ti S t / Infected species Chmotactic Bacteria Symptoms/ Diseases Various crops Dickeya dadantii Necrosis p (potato, chicory) y Mammals (inc. humans) Spirochetes Lyme disease, l f relapsing fever, syphilis Fish humans Vibrio Various infections Fish, humans Vibrio Various infections Humans Helicobacter pylori Inflammation, Ulcers
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001 MGSYHHHHHHSSGLVPRGSHMKSGTNVDQIVERVKEASSQIGETLENIRSIEKLIQNIMR 001 MGSYHHHHHHSSGLVPRGSHMKSGTNVDQIVERVKEASSQIGETLENIRSIEKLIQNIMR 001 ************************************************************ 061 IARETNILALNATIEAARAGEAGKGFMLVANEVQNLSNETNEVTKQIVEKAREILESSQR 061 IARETNILALNATIEAARAGEAGKGFMIVANEVQNLSNETNEVTKQIVEKAREILESSQR 061 *************************** ******************************** 121 SLENLEFMANLFETVGKT 121 SLENLEFMANLFETVGKT 121 ****************** Top- Top- Mutant Mutant Bottom Bottom Wildtype Wildtype Bottom Bottom- Wildtype Wildtype Single amino-acid mutation. Single amino-acid mutation.
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