Two-color sum-frequency generation spectroscopy (2C-SFG) is used to probe the molecular and electronic properties of an adsorbed layer of the green fluorescent protein mutant 2 (GFPmut2) on a platinum (111) substrate. First, the spectroscopic measurements, performed under different polarization combinations, and atomic force microscopy (AFM) show that the GFPmut2 proteins form a fairly ordered monolayer on the platinum surface. Next, the nonlinear spectroscopic data provide evidence of particular coupling phenomena between the GFPmut2 vibrational and electronic properties. This is revealed by the occurrence of two doubly resonant sum-frequency generation processes for molecules having both their Raman and infrared transition moments in a direction perpendicular to the sample plane. Finally, our 2C-SFG analysis reveals two electronic transitions corresponding to the absorption and fluorescence energy levels which are related to two different GFPmut2 conformations: the B (anionic) and I forms, respectively. Their observation and wavelength positions attest the keeping of the GFPmut2 electronic properties upon adsorption on the metallic surface.

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Two families of bacterial serine enzymes recognize penicillin and other ␤-lactam antibiotics and form a superfamily of evolutionarily related proteins named serine penicillin-recognizing enzymes (PREs) (21). The first family contains the D-alanyl-D-alanine peptidases (DD-transpeptidases) involved in the biosynthesis or in the control of the cross-linking of the peptidoglycan, the main constituent of the bacterial cell wall. DD-peptidases are inactivated by ␤-lactam antibiotics, as a result of the acylation of their active serine by the antibiotic, which yields a very stable covalent adduct (Fig. 1). Because of these properties, these enzymes are also referred to as penicillin-binding proteins (PBPs) (10). The members of the second family, the serine ␤-lactamases are subdivided into three classes (A, C, and D) and efficiently hydrolyze the amide bond of the ␤-lactam nucleus, to yield products devoid of antibiotic activity (12, 31). For serine ␤-lactamases, the catalytic pathway involves the formation of an acyl-enzyme similar to that obtained in the interaction of the ␤-lactam antibiotic with the DD-peptidases, but in the case of ␤-lactamases the acyl-enzyme is very unstable. Although one might think that they would be very different, the two families of enzymes share many biochemical characteristics and very similar three-dimensional (3D) structures (17,22,33,36). Along their primary structures, three conserved peptide sequences, important for recognition of the substrate or catalysis, have been identified by comparison of their 3D structures (Table 1). Sequence identities between the class D and the class A and C enzymes are on average 16% (29). Nevertheless, these proteins are thought to have a common evolutionary history (15,21,24).By database comparison, three open reading frames, penP, ybbE, and ybxI, have been found in the B. subtilis 168 genome which encode putative ␤-lactamases. PenP has been overproduced and characterized as a class A ␤-lactamase (J. M. van Dijl, unpublished data), whereas YbbE remains uncharacterized, and YbxI has been postulated to be a class D ␤-lactamase because its primary structure exhibits a high similarity with the members of this group of serine ␤-lactamases. A phylogenetic tree was constructed by Barlow and Hall (3), in which the ybxI nucleotide sequence was referred to as B. subtilis z99105. No promoter sequence was identified upstream of the coding sequences for these three genes, in agreement with the fact that no ␤-lactamase activity was detected in growing B. subtilis 168 cells or in the supernatant. To determine if YbxI is a class D ␤-lactamase, it was overproduced in the periplasm of Escherichia coli, purified to homogeneity, and analyzed for its capacity to hydrolyze ␤-lactam antibiotics and DD-peptidase substrates. YbxI was shown to be a ␤-lactamase of very low activity which is not inactivated by clavulanic acid. Indeed, the recombinant protein interacts with several ␤-lactam antibiotics and can hydrolyze some of them but does not hydrolyze the peptides cleaved by DD...