Structural Changes Revealed by Fourier Transform Infrared and Circular Dichroism Spectroscopic Analyses Underlie tNOX Periodic Oscillations

Abstract

A recurring pattern of spectral changes indicative of periodic changes in the proportion of β-structure and a-helix of a recombinant ECTO-NOX fusion protein of tNOX, with a cellulose binding domain peptide, was demonstrated by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic analyses. The pattern of structural changes correlated with oscillatory patterns of enzymatic activities exhibited by the protein previously interpreted as indicative of a clock function. The pattern consisted of a repeating pattern of oscillations with a period length of 21 min with five maxima (two separated by 5 min and 3 separated by 4 to 4.5 min) within each 21 min repeat. Oscillatory patterns were not obvious in comparable FTIR or CD spectra of albumin, ribonuclease or concanavalin A. The period length was constant at 5, 15, 25, 35 and 45°C (temperature compensated) and oscillations occurred independently of substrate presence. Spectra obtained in deuterium oxide yielded a longer period length of 26 min both for oscillations in enzymatic activity and absorbance ratios determined by FTIR. Taken together the findings suggest that the regular patterns of oscillations exhibited by the ECTO-NOX proteins are accompanied by recurrent global changes in the conformation of the protein backbone that directly modulate enzymatic activity.

A role for copper in biological time-keeping

“…The rate of enlargement of plant, animal and bacterial cells also oscillates with a period of 24 min [12][13][14]29,30]. The 24 min periodic behavior is exhibited by pure recombinant tNOX protein and is accompanied by a recurring pattern of amide I/amide II FTIR and CD spectral changes suggestive of a-helix-b-sheet transformations [31]. ECTO-NOX proteins share many characteristics with prions [32,33] and the a-amyloid forming proteins of neurodegenerative diseases [34].…”

“…The conformational changes associated with time keeping [31] observed by FTIR and CD would appear to be coupled to the alterations in the copper ligands related to changes in redox state. The copper of tNOX is apparently ligated to histidines of the H546 VH copper site plus the downstream H562.…”

Ultradian oscillators of the circadian clock in <i>Saccharomyces</i> <i>cerevisiae</i>

“…When mammalian ENOX was assayed in heavy water, it exhibited a period length of about 30 min [25]. Heat-inactivated yeast assayed in heavy water yielded period lengths of approximately 30 …”