Pressure-induced conformational changes in a human Bence-Jones protein (Mcg)

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Abstract
The effect of high static pressures on the internal structure of the [human] Ig L chain (Bence-Jones) dimer was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (< 2 kbar), with a standard vol change of -93 ml/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis-(8-anilinonaphthalene-1 sulfonate) (bis-ANS), rhodamine 123 and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the M dimer exhibited a small decrease in affinity for bis-ANS (standard vol change .apprx. 5.9 ml/mol). At 3 kbar the binding activity increased by > 250-fold (volume change-144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures > 1.5 kbar (standard vol change -23 ml/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard vol change -55 ml/mol), but the protein was partially denatured at pressures > 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds. The complementarity between this binding region and a suitable ligand can be improved by the imposition of external pressures far below those causing general disruption of the protein structure.