In this article we characterize, by means of the perturbed matrix method, the response of the electronic states of a chemical system to the perturbing environment. In the theory section we describe in detail the basic derivations and implications of the method, extending its theoretical framework to treat possible excitonic effects, and we show how to characterize the perturbed electronic states. Finally, by using a set of chemical systems interacting with complex atomic-molecular environments, we describe the nature and general features of the electronic state mixing and transitions as caused by atomic and molecular interactions.

Summary: FlexServ is a web-based tool for the analysis of protein flexibility. The server incorporates powerful protocols for the coarse-grained determination of protein dynamics using different versions of Normal Mode Analysis (NMA), Brownian dynamics (BD) and Discrete Dynamics (DMD). It can also analyze user provided trajectories. The server allows a complete analysis of flexibility using a large variety of metrics, including basic geometrical analysis, B-factors, essential dynamics, stiffness analysis, collectivity measures, Lindemann's indexes, residue correlation, chain-correlations, dynamic domain determination, hinge point detections, etc. Data is presented through a web interface as plain text, 2D and 3D graphics. Availability:http://mmb.pcb.ub.es/FlexServ; http://www.inab.org Contact:modesto@mmb.pcb.ub.es or gelpi@mmb.pcb.ub.es Supplementary information: Additional information and methodology details can be found at http://mmb.pcb.ub.es/FlexServ/help.

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein–DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo .