Site-selective dynamics of azidolysozyme.

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The spectroscopic response of and structural dynamics around all azido-modified alanine residues (AlaN3) in lysozyme are characterized. It is found that AlaN3 is a positionally sensitive probe for the local dynamics, covering a frequency range of ∼15 cm-1 for the center frequency of the line shape. This is consistent with findings from selective replacements of amino acids in PDZ2, which reported a frequency span of ∼10 cm-1 for replacements of Val, Ala, or Glu by azidohomoalanine. For the frequency fluctuation correlation functions, the long-time decay constants τ2 range from ∼1 to ∼10 ps, which compares with experimentally measured correlation times of 3 ps. Attaching azide to alanine residues can yield dynamics that decays to zero on the few ps time scale (i.e., static component Δ0 ∼ 0 ps-1) or to a remaining, static contribution of ∼0.5 ps-1 (corresponding to 2.5 cm-1), depending on the local environment on the 10 ps time scale. The magnitude of the static component correlates qualitatively with the degree of hydration of the spectroscopic probe. Although attaching azide to alanine residues is found to be structurally minimally invasive with respect to the overall protein structure, analysis of the local hydrophobicity indicates that the hydration around the modification site differs for modified and unmodified alanine residues, respectively.