Elimination of the Q R bridge significantly

Elimination of the Q234–R244 bridge significantly affected the protein sr9011 synthesis but had little effect on its activity at a permissive temperature, which may seem contrary to the known functional importance of this motif in Fpg. However, since the mutants fully or partially retained the bound zinc (Table 2), the structure of the finger itself was presumably intact. In MD runs, the mutations in the Q234–R244 bridge did not affect the solvent-accessible area and orientation of the finger relative to the rest of the protein and even decreased the r.m.s.f. values of several residues in the zinc finger. Therefore, the main function of the Q234–R244 bridge seems to be the thermodynamic stabilization of the C-terminal domain of the protein rather than maintaining the conformation of the zinc finger. Molecular dynamics of Fpg mutants studied here revealed a number of hydrogen bonds preferentially formed in either active or inactive Fpg variants (Supplementary Table 1). Notably, the majority of these bonds was located either in the zinc finger or in the interdomain linker and the immediately adjacent part of the C-terminal domain, or in the zinc finger. The latter region was identified before in L. lactis Fpg as forming different bonds when the enzyme binds a good substrate (oxoG:C) vs poor substrate (oxoG:A), despite being far from the DNA-binding groove [25]. The possibility of this region influencing the linker dynamics and having a synergistic effect with the R54–E131 bridge warrants further investigation.
Introduction Over the past few decades, many efforts have been directed to design innovative and efficient optical biosensors based on plasmonic transducers, such as surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR) [[1], [2], [3], [4]]. In particular, SERS has been proven to be a powerful analytical technique and widely applied in chemical [5,6], environmental [7,8], and biomedical [9,10] fields owing to its significant advantages including nondestructive measurement, resistance to photobleaching, and narrow emission peaks for spectral multiplexing. However, applications of SERS are often associated with qualitative [11] and semiquantitative [12] assay, which might be attributed to the dissatisfactory reproducibility and stability of the measurements resulted from the non-uniform SERS substrates, the wide range of enhancement factors (EFs), and the discrepant distance of inter-coupling between adjacent nanoparticles. Thus, it is urgent to develop the quantitative SERS techniques to facilitate the practical applications in chemical and biomolecular analysis. SERS, as we know, enables the detection of very-low-concentration of analytes even single molecules, which is related to the localized electromagnetic field enhancement of plasmonic nanoparticles [13]. It is common in bioanalytical SERS that target-induced aggregation or deaggregation of plasmonic nanoparticles is employed to generate or vanish of hot spots, thus SERS signal will turn on and off, respectively. For example, Wang et al. developed a simple SERS platform for detecting heparin based on antiaggregation of 4-mercaptopyridine functionalized Ag nanoparticles (4-MPY-AuNPs) upon the presence of protamine [14]. In their study, protamine was employed as a medium for inducing the aggregation of negatively charged 4-MPY-AuNPs via electrostatic interaction. Zong et al. reported a novel SERS sensing platform for ultrasensitive detection of telomerase using telomeric elongation controlled SERS effect [15]. Although the facility and low cost of these methods, the addition of aggregation agents might result in the irregular distribution of the aggregates, and the formation of small aggregates produced by the nonspecific absorption effects, which strongly influences the stability and reproducibility of the SERS signal. Lately, the self-assembly plasmonic nanostructures with well-defined architectures and effective coupling have drawn considerable interest in SERS detection because of their remarkably enhancement, and excellent reproducibility and controllability. Tanwar et al. demonstrated the synthesis of Au nanostar dimers with tunable interparticle gap and controlled stoichiometry assembled on DNA origami that can be used as active plasmonic nanoplatforms for single-molecule SERS sensing [16]. Tian et al. fabricated a novel plasmonic heterodimer nanostructure with controllable self-assembled hot spots by the conjugation of individual Au@Ag core-shell nanocubes and varisized gold nanospheres via the biotin-streptavidin interaction [17]. Although these methods have ability to detect target with exciting sensitivity, the preparation of SERS substrates is extremely complicated and the different batches of substrates is not exactly the same, which is detrimental to the practicability and repeatability of the methods. Previous studies have demonstrated that the distance between plasmonic nanoparticles significantly impact electromagnetic (EM) enhancement [18]. The EM enhancement decays drastically as the increase of the distance between the plasmonic nanoparticles [19]. The wide distribution of EFs values can result in a poor reproducibility of the SERS signal [20]. Therefore, it remains a major challenge for quantitative SERS to lightly and repeatedly acquire hot spots with uniformly distribution and narrow EFs to improve SERS detection of analyte.