Membrane-induced changes in the holomyoglobin tertiary structure: interplay with function

Abstract

The effect of anionic phospholipid membranes on holomyoglobin (holoMb) conformation and deoxygenation was studied. HoloMb structural changes and behavior in the presence of membranes were monitored by a variety of techniques including far UV and near UV circular dichroism, tryptophan (Trp) fluorescence, absorbance in the Soret region, differential scanning calorimetry, (1)H-NMR spectroscopy, size exclusion chromatography, and macroscopic diffusion. Kinetics of deoxygenation was monitored by absorption at 581 nm. The results gave evidence that proximity to a negatively charged membrane surface can cause destabilization of the structure of holomyoglobin, which delivers oxygen (O2) to mitochondria. It was shown that holoMb undergoes the native-to-intermediate-state transition in the presence of anionic phospholipid membranes at neutral pH, and that in this state it is able to interact with the membranes. When in the intermediate state, holoMb loses its rigid tertiary structure but preserves a pronounced secondary one. The presence of anionic phospholipid membranes substantially accelerates the process of deoxygenation. A possible functional role of the more flexible protein structure acquired in immediate proximity to the membrane surface is discussed.

How membrane surface affects protein structure

“…13 2014 Thermal melting is typical of both forms. In the presence of phospholipids, the mentioned temperature transition disappears, but for apoMb this occurs at PhL/Pr ratio of 25 : 1, while for holoMb this ratio is 200 : 1 [60]. The absence of the melting peak is evidence of disturbance of the tight packing of side groups.…”

“…An increase in the concentration of phos pholipids leads to a decrease in the molar ellipticity value, retaining the shape of the spectra, which shows that the number of protein molecules with a rigid tertiary struc ture is reduced. With molar ratios of PhL/Pr = 200 : 1 at pH 7.2 and 25 : 1 at pH 6.2, all specific features of the spectrum disappear, which is evidence of the loss of tight packing of side groups in the presence of a high content of negatively charged phospholipid vesicles [60].…”

“…Detailed studies of holoMb and apoMb have been done previously [60,61]. The changes in the shape and intensity of CD spectra in the near UV region yield information on the disturbance of the protein tertiary structure in the presence of nega tively charged vesicles from POPG, whose molar ratios (PhL/Pr) were varied from 25 : 1 to 200 : 1 at pH 7.2.…”

“…With molar ratio PhL/Pr = 25 : 1 (pH 7.2) and the use of the gel filtration chromatogra phy, a peak is observed in the range of the native protein elution, i.e. the main part (~75%) of the protein remains unbound, though about a quarter of the protein is eluted in the peak corresponding to phospholipid vesicles [60]. At higher PhL/Pr molar ratios, the fraction of free pro tein decreases simultaneously with an increase in the portion of the protein bound to the phospholipid vesicles.…”

“…The extent of the protein binding to the vesicles correlates with the portion of the denatured protein tested by CD in the near UV region [60]. As known, in the N state the protein does not inter act with membranes [3,5]; that is why only the confor mational transition of myoglobin to the intermediate state with a larger hydrophobic surface results in the bind ing of the protein to the vesicles.…”