Structural deformations of lipid hybrid bilayer membranes induced by signal peptideless (SPL) proteins have been studied for the first time using the inherently surface specific nonlinear optical technique of sum frequency generation vibrational spectroscopy. Specifically, deformations of 1,2-distearoylphosphatidylglycerol (DSPG) membranes induced by interaction with FGF-1, a SPL protein which is released as a function of cellular stress through a nonclassical pathway, have been investigated. FGF-1 was found to induce lipid alkyl chain deformations in previously highly ordered DSPG membranes at the extremely low concentration of 1 nM at 60degreesC. The deformation process was shown to exhibit a degree of reversibility upon removal of the protein by rinsing with buffer solution.