Lack of Gross Protein Structure Changes in the Working Cycle of (Na⁺, K⁺)‐Dependent Adenosinetriphosphatase

Abstract

Infrared and tryptophan fluorescence spectra of practically all sufficiently stable functional con~plexes of a highly purified preparation of membrane-bound (Na', K +)-dependent ATPase have been measured. The formation of any functional complex was not accompanied by any considerable change of either shape or position of the tryptophan fluorescence spectrum. Only in the presence of adenine nucleotides was there a small decrease of fluorescence intensity (by 5 -8 %), which apparently results from a change of the sample light scattering. Analysis of the results obtained leads to the conclusion that the environment of no more than one or a few tryptophan residues may differ in all the (Na', K+)-ATPase complexes studies. A comparison of infrared protein spectra in the region of amide I band showed that at any wavenumber the differences between them did not exceed 3 % of the maximum absorption. This means that no more than 3 % of protein peptide groups can change their conformation upon transition between the enzyme functional states. These results, obtained by two independent techniques, allow us to conclude that even if changes of the internal protein structure occur during the working cycle of this transport system, they have an extremely local character.

Fluorescence spectroscopic studies on equilibrium dipole-relaxational dynamics of Na,K-ATPase

“…The small difference of the fluorescence spectra between form E, and Et of the enzyme that we have found has been reported also by Chetverin and coworkers [31], who calculated difference spectra for both conformations. An explanation for the shift of the maximum indepen-A.P.…”

“…If we assume that there is no excited-state energy transfer between tryptophanes, the fluorescence spectrum of the protein should be the sum of the contributions of the individual tryptophanes, and it can be expected that the spectrum is broader than for a single-tryptophan protein, because the tryptophanes emit at different wavelengths. However, the width at half-maximum intensity of the fluorescence spectrum for Na,K-ATPase is 59 nm [31], which is not substantially broader than the 143 spectra of single-tryptophan proteins emitting at these wavelengths [32]. According to Burstein's classification the spectrum of an individual tryptophan residue emitting at 340-342 nm should have a half-width of 53-55 nm [32].…”

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy

“…Structural changes during Tris ---> K * --Na* buffer exchange Secondary structural changes of the Na*7K*-ATPase as revealed by the amide I, I' band are not easy to be detected by IR spectroscopy of a membrane fragment suspension [28][29][30] as well as by IR-ATR spectroscopy of adsorbed fragments (this paper). Although the z(NH) band of the non exchanging core of the protein (about 247o of the amino acids) indicated a structural change upon buffer exchange, there is no evidence for such a conformational transition from the overall dichroic ratio of the amide I, I' band (cf.…”

“…In the regions of strong liquid DrO absorption (z(DrO), =2500 cm-1; ö(DrO), =1200 cm-r) there is a slight solvent overcompensation which results from the reduced effective thickness of the aqueous medium due to membrane fragment adsorption. Prominent protein absorption bands are [78]: z(NH), NH-stretching at = 3300 cm-1 which reflects the hard core of the protein [28][29][30]; amide I, I' ( = 80Vo C:Ostretching of amide group, the apostrophe denoting the vibration of the N-deuterated group) at =1645 cm-1; 2,,(COO-) (antisymmetric CO; stretching of gluta- Ir mate, aspartate) at = 1580 cm-r; amide II (=607o NH-bending) at = 1540 cm-r; amide II' (partly NDbending) at = 1460 cm-r; z. (COO-) (symmetric COf stretching of glutamate and aspartate) at = 1400 cm-t.…”

“…While such studies in principle can yield information on the presence of a-helical, ß, and random-coil structures in the protein, the interpretation of circulardichroism spectra of Na*7K*-ATPase preparations is difficult due to scattering and absorption-flattening effects125,27]. Chetverin, Brazhnikov and co-workers have carried out infrared spectroscopic studies of suspensions of particulate Na*/K*-ATPase in DrO [28][29][30]. From a line-shape analysis of the amide I band, they estimated that about 22Vo of the peptide bonds are involved in a-helices and 22Vo in B-pleated sheets [30].…”