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B.J. Glasgow, O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov; Distance Measurements Between Interstrand Loops EF and CD of Tear Lipocalin Support a Mechanism of pH Regulated Access to the Calyx . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3888.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Tear lipocalin (TL), the major lipid binding protein in tears releases lipid ligands in an acidic environment. A 3D model based on secondary structure assignment in solution shows that interstrand loops overhang the calyx entrance (Gasymov et al., Biochemistry 2001;40,14754–62). We tested the hypothesis that loops E–F and C–D form a pH triggered gate that regulates access to the binding cavity. Methods: Site–directed mutagenesis using sequential PCR steps, plasmid construction, expression, purification, and analysis of mutant proteins by CD spectroscopy were performed as described for TL (Ibid). For distance measurements, two mutants of TL were constructed with Trp and Cys substitutions on loops C–D and E–F, respectively (Trp62/Cys82, Trp62/Cys81). Cys residues of both mutant proteins were labeled with MTS–Dansyl. Fluorescent emission spectra were measured, λex= 295 nm, in 10 mM Na–phosphate (pH 8.5–5.5) or 30mM Na–citrate (pH 4.0–3.0). Quenching experiments were performed by progressive addition of aliquots of acrylamide to Trp mutants. Data were fit to the Stern–Volmer equation to find collision rate constants (kq). Time–resolved intensity decay data were obtained under the same pH conditions using the phase/modulation frequency domain. Data analyses were performed with nonlinear least–squares fitting. Interstrand loop distances were calculated from the lifetime of the donor (Trp) with (τDA) and without (τD) the acceptor (fluorescent label) relating calculated energy transfer efficiency (E=1– τDA/τD) to distance between the donor and acceptor (r) by E=R06 /R06+r6 , R0 =Förster distance. Results: CD spectroscopy showed that the intrinsic secondary structure in TL was not perturbed by the mutations. At pH 7 the interstrand loop distances (between Trp and the dansyl label) were 23.4 and 31.5 Å for Trp62/Cys82 and Trp62/Cys81, respectively. At pH 3, these distances decreased to 21.4 and 23.4 Å. The residues on both loops showed blue–shifted λmax and reduced kq at pH 3 when compared to these parameters at pH 7 indicative of an environment with less solvent exposure. Conclusions: The mutants of TL provide information about pH driven dynamic changes in the distances between loops C–D and E–F. The closure of interstrand loops C–D and E–F is triggered by a low pH. The accompanying blue shift of λmax and reduced kq at low pH is consistent with closer apposition of the loops. The data support a model in which the interstrand loops act as gates to regulate access of ligands to the lipocalin cavity that loses binding affinity to fatty acids with decreasing pH.
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