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. 1991 Mar;59(3):654–669. doi: 10.1016/S0006-3495(91)82281-0

Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy.

T C Squier 1, J E Mahaney 1, J J Yin 1, C S Lai 1, J R Lakowicz 1
PMCID: PMC1281230  PMID: 1646658

Abstract

We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed.

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Selected References

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