Contents lists available at ScienceDirect Biophysical Chemistry journal homepage: www.elsevier.com/locate/biophyschem Small phospho-donors phosphorylate MorR without inducing protein conformational changes Nathália S.S. Castro a, ⁎ , César A.T. Laia b , Biplab K. Maiti a , Nuno M.F.S.A. Cerqueira b , Isabel Moura a , Marta S.P. Carepo a a LAQV-REQUIMTE, Departamento de Química, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal HIGHLIGHTS • Phosphoramidate and acetylpho- sphate phosphorylate MorR in several aminoacid residues • Phosphorylation does not induce MorR oligomerization. • MorR-Tryptophan emission is quen- ched in the presence of phosphor- amidate. • Phosphoramidate is energetic favor- able to phosphorylation. GRAPHICAL ABSTRACT ARTICLE INFO Keywords: Two-component system MorR NtrC Phosphorylation Phosphoramidate Acetylphosphate ABSTRACT Phosphorylation is an essential mechanism of protein control and plays an important role in biology. The two- component system (TCS) is a bacterial regulation mechanism mediated by a response regulator (RR) protein and a kinase protein, which synchronize the regulatory circuit according to the environment. Phosphorylation is a key element in TCS function as it controls RR activity. In the present study, we characterize the behavior of MorR, an RR associated with Mo homeostasis, upon acetylphosphate and phosphoramidate treatment in vitro. Our results show that MorR was phosphorylated by both phospho-donors. Fluorescence experiments showed that MorR tryptophan emission is quenched by phosphoramidate. Furthermore, theoretical and computational results demonstrate that phosphorylation by phosphoramidate is more favorable than that by acetylphosphate. In conclusion, phosphorylated MorR is a monomeric protein and phosphorylation does not appear to induce ob- servable conformational changes in the protein structure. 1. Introduction The two-component system (TCS) is a dynamic biological circuit formed by a histidine kinase (HK) protein and a response regulator (RR) protein that typically acts as a transcriptional regulator [1, 2]. In bac- teria, extracellular signals are converted to a cellular response via a general, reversible phospho-transfer from the HK to the RR and the management of crosstalk fidelity between these two proteins [3–6]. Among TCS RRs, phosphorylation is the main post-translational signal capable of inducing conformational changes and protein output responses. RR acetylation has also been reported, but only a few ex- amples are available, such as CheY, an RR of the E. coli chemotaxis system, and RcsB, an RR involved in the regulation of more than 100 bacterial genes [7–11]. A variety of analytical tools have been utilized https://doi.org/10.1016/j.bpc.2018.05.009 Received 14 February 2018; Received in revised form 10 May 2018; Accepted 29 May 2018 ⁎ Corresponding author. E-mail address: n.castro@campus.fct.unl.pt (N.S.S. Castro). Biophysical Chemistry 240 (2018) 25–33 Available online 31 May 2018 0301-4622/ © 2018 Elsevier B.V. All rights reserved. T