Amino acid selective labeling and unlabeling for protein resonance assignments.

Structural characterization of proteins by NMR spectroscopy begins with the process of sequence specific resonance assignments in which the (1)H, (13)C and (15)N chemical shifts of all backbone and side-chain nuclei in the polypeptide are assigned. This process requires different isotope labeled forms of the protein together with specific experiments for establishing the sequential connectivity between the neighboring amino acid residues. In the case of spectral overlap, it is useful to identify spin systems corresponding to the different amino acid types selectively. With isotope labeling this can be achieved in two ways: (i) amino acid selective labeling or (ii) amino acid selective 'unlabeling'. This chapter describes both these methods with more emphasis on selective unlabeling describing the various practical aspects. The recent developments involving combinatorial selective labeling and unlabeling are also discussed.

Amino acid selective unlabeling for sequence specific resonance assignments in proteins.

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective 'unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly (13)C/(15)N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {(12)CO( i )-(15)N( i+1)}-filtered HSQC, which aids in linking the (1)H(N)/(15)N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to (2)H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of (14)N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

Spontaneous and reversible self-assembly of a polypeptide fragment of insulin-like growth factor binding protein-2 into fluorescent nanotubular structures.

In this communication, we report the spontaneous and reversible in vitro self-assembly of a polypeptide fragment derived from the C-terminal domain of Insulin-like Growth Factor Binding Protein (IGFBP-2) into soluble nanotubular structures several micrometres long via a mechanism involving inter-molecular disulfide bonds and exhibiting enhanced fluorescence.