sequential-assignment - Sequential Assignment Strategies in...

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Sequential Assignment Strategies in Proteins
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NMR assignments • in order to determine a structure by traditional, NOE-based 1 H- 1 H distance-based methods, the chemical shifts of the individual 1 H nuclei must be known • older methods based on homonuclear ( 1 H) experiments only, whereas newer methods based on heteronuclear / triple resonance experiments • triple resonance techniques generally require 1 H, 13 C and 15 N chemical shift assignments in a protein • chemical shifts so obtained are useful for predicting secondary structure of proteins including main chain φ and ψ angles
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1 H methods: The Sequential Assignment Procedure • due to Wüthrich and coworkers (early/mid 1980’s) “1. Identification of amino acid side chain spin systems. 2. Identification of neighboring residues in the amino acid sequence. 3. Suitable combinations of the results from 1 and 2 which provides individual resonance assignments in the primary structure of the protein.” K. Wüthrich 1983 Biopolymers 22 , 131-138 1). Use J correlation spectroscopy (COSY, TOCSY) to identify individual spin systems (amino acids) 2). Use NOE spectroscopy (NOESY) to link amino acids Book : “NMR of Proteins and Nucleic Acids”, Kurt Wüthrich, 1986 (Wiley)
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1 H methods: The Sequential Assignment Procedure 1). Identify amino acid spin systems using COSY and TOCSY • Spin systems are classified based on the spins that can be correlated in COSY spectra, and then TOCSY spectra -remember, there is no way to connect amino acids in a protein by COSY or TOCSY -a COSY or TOCSY spectrum of a protein is comprised of the individual spectra of the amino acids in the protein • the individual spin systems are identified as to amino acid type -how? Based on “amino acid specific patterns” of COSY and TOCSY cross peaks Asp Val
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More examples of COSY patterns
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1 H chemical shift information 1 H chemical shift information is also very useful in assisting to identify spin systems -shown are distributions of 1 H chemical shifts for amino acids in proteins Wishart et al. (1991) J. Mol. Biol. 222 , 311-333
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1 H methods: The Sequential Assignment Procedure 2). Link identified amino acids using NOESY data • critical observation: it is virtually impossible to align the main chain atoms of two adjacent amino acid residues in a protein so that there is
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This note was uploaded on 11/07/2011 for the course CHEM 8853R taught by Professor Gelbaum during the Fall '11 term at Georgia Institute of Technology.

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sequential-assignment - Sequential Assignment Strategies in...

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