Methods for determining protein structure Sequence: Edman degradation

Methods for determining protein structure  Sequence:  Edman degradation

Methods for determining protein structure Sequence: Edman degradation Mass spectrometry Secondary structure: Circular Dichroism FTIR Tertiary, quaternary structure: NMR

X-ray crystallography Protein sequencing approaches depend on what is known and what is the goal Protein is unknown, from organism with no DNA sequence information starting from scratch

Purify protein & separate chains (if multimer) Fragment and sequence each chain Fragment differently and sequence Reassemble sequence based on overlapping fragments Protein is unknown or known, and comes from an organism with known DNA sequence Purify protein (& separate chains) Fragment chain(s) and sequence or measure mass Use sequence database to reassemble sequence

Protein sequencing from scratch Step 0: Purify the protein Step 1: Separate the chains (if multimeric) If needed, reduce disulfides and block free thiols Protein sequencing from scratch Step 0: Purify the protein Step 1: Separate the chains (if multimeric) Step 2: Fragment each polypeptide Enzymatically, with endopeptidase, chemically (e.g. with cyanogen bromide), or physically (e.g. through

collision in MS) Step 2: Fragment each polypeptide Cyanogen bromide (CNBr): Rn-1 = Met Protein sequencing from scratch

Step 0: Purify the protein Step 1: Separate the chains (if multimeric) Step 2: Fragment each polypeptide Step 3: Sequence the fragments Via, e.g., Edman degradation or Mass spectrometry Sequence peptides with mass spectrometry (MS/MS) MS cleavage occurs mainly at peptide

bonds, and charge is retained in one product Protein sequencing from scratch Step 0: Purify the protein Step 1: Separate the chains (if multimeric)

Step 2: Fragment each polypeptide Step 3: Sequence the fragments Step 4: Reconstruct the sequence Protein sequencing approaches depend on what is known and what is the goal Protein is unknown, from organism with no DNA sequence information starting from scratch

Purify protein & separate chains (if multimer) Fragment and sequence each chain Fragment differently and sequence Reassemble sequence based on overlapping fragments Protein is unknown or known, and comes from an organism with known DNA sequence Purify protein (& separate chains) Fragment chain(s) and sequence or measure mass

Use sequence database to reassemble sequence There are different approaches for using mass spectrometry to sequence a protein Bottom-Up Proteomics Fragment protein (e.g. enzymatically) and separate fragments Ionize fragments, trap in the spectrometer, and measure m/z Select one m/z peak and fragment (e.g. by collision) Measure m/z of the smaller fragments and use a database to match the peaks to known sequences

There are different approaches for using mass spectrometry to sequence a protein Top-Down Proteomics Ionize whole protein(s), trap in the spectrometer, and measure m/z Use the instrument to select one m/z peak and fragment the protein (e.g. by collision) Measure m/z ratios of the fragments and use a database to match the peaks to known sequences OR Select a peak and fragment again, then match to sequence

(Selection and fragmentation may be repeated over and over) In shotgun proteomics, mass spec. is used to sequence mixtures of proteins Mixture of many proteins Enzymatic digest Mixture of

peptides from different proteins Separation of peptides, Ionization in MS, Fragmentation Matched sequences Submit peaks to database

Methods for determining protein structure Sequence: Edman degradation Mass spectrometry Secondary structure: Circular Dichroism FTIR Tertiary, quaternary structure: NMR

X-ray crystallography Circular dichroism (CD) measures amide absorption of circularly polarized UV light Ellipticity (De) is the difference in absorption of left-handed and righthanded circularly polarized light Different secondary structures show different patterns of ellipticity

Proteins CD spectrum is deconvoluted to estimate fractional contribution of helix, sheet, turn, and coil Proteins with different compositions of 2 structure give different CD spectra Fourier transform infrared (FTIR) spectra show amide absorption of infrared light

Peak frequencies show bond stretching and bending, which vary with protein conformation C=O stretching frequency of amide I band correlates with secondary structure Proteins FTIR spectrum is deconvoluted to estimate fractional

contribution of helix, sheet, and coil Methods for determining protein structure Sequence: Edman degradation Mass spectrometry Secondary structure: Circular Dichroism FTIR

Tertiary, quaternary structure: NMR X-ray crystallography Proteins have too many protons to be resolved by one-dimensional NMR 2D NMR separates proton peaks and can reveal approximate distances between nearby atoms

a b c d Cross-peaks indicate protons are within 5 of each other NMR-derived distance constraints are used to calculate likely protein conformations

X-ray crystallography reveals the layout of repeating electron density Diffracted X-rays Data processing X-rays Protein crystal

Diffraction pattern Electron density map Electron density map allows for positioning of protein atoms, revealing structure

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