Metadynamics is a powerful technique that has been successfully exploited to explore the multidimensional free energy surface of complex polyatomic systems and predict transition mechanisms in very different fields, ranging from chemistry and solid-state physics to biophysics. We here derive an explicit expression for the accuracy of the methodology and provide a way to choose the parameters of the method in order to optimize its performance.

This paper is concerned with the dynamics of water around a small globular protein. Dipolar second-rank relaxation time and diffusion properties of surface water were computed by extensive molecular dynamics simulations of lysozyme in water which lasted a total of 28 ns. Our results indicate that the rotational relaxation of water in the vicinity of lysozyme is 3-7 times slower than that in the bulk depending on how the hydration shell is defined in the calculation. We have also verified that the dynamics of water translational diffusion in the vicinity of lysozyme have retardations similar to rotational relaxation. This is a common assumption in nuclear magnetic relaxation dispersion (NMRD) studies to derive residence times. In contrast to bulk water dynamics, surface water is in a dispersive diffusion regime or subdiffusion. Very good agreement of dipolar second-rank relaxation time with NMRD estimates is obtained by using appropriate dimensions of the hydration shell. Although our computed second-rank dipolar retardations are independent of the water model, SPC/E describes more realistically the time scale of the water dynamics around lysozyme than does TIP3P.

Gram-negative bacteria and their complex cell envelope comprising an outer and inner membrane are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular penetration of small molecules, including nutrients and antibacterial agents. The synergistic action between relatively slow porin-mediated passive uptake across the outer membrane and active efflux transporters in the inner membrane creates a permeability barrier that reinforces the enzymatic modification barrier, which efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in small molecule translocation in Enterobacteriaceae and consider the crucial role of porins in antibiotic resistance. Commented [w1]: Is this specification necessary here?, in my opinion it deviates, better to put later… Commented [JP2]: Editor request... porins represent the preferred route for the entry of β-lactams, including cephalosporins, penicillins and carbapenems 14-16. The clinical relevance of membrane-associated mechanisms (MAMs) of resistance (i.e. porin defects and/or overexpression of multidrug efflux pumps) has been well established for these antibiotics. The Influx and Efflux rates control the internal concentration of antibiotics and represent the first lane (mechanical barrier) protecting the bacterial cells against therapeutic treatment 1-3,6. Consequently, studies on bacterial porins are receiving a renewed interest due to their key role in the bacterial susceptibility towards clinically used antibiotics. In combination with the expression of antibiotic-modifying enzymes expressed in the periplasm (e.g. β-lactamases), porins play a key role in β-lactam resistance 4,17. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in antibiotic translocation in Enterobacteriaceae. We explore structural aspects and the insights gained into permeation and the pore translocation process, the regulation of porin expression as well as the role of porins in the emergence of antibiotic susceptibility. Enterobacterial general porins Structural aspects The crystal structures of a general porin from Rhodobacter capsulatus 18 , the OmpF and PhoE porins from E. coli 19 and other E. coli OmpF structures including mutants 20,21 were the first to be solved. Only a limited number of other enterobacterial porin structures have been reported, i.e. E. coli OmpC, K. pneumoniae OmpK36 and Salmonella typhi OmpF 22-24. The lack of data has hindered attempts to relate structure to function. Recently, the structures of two porins from P. stuartii as well as the structures of the OmpF and OmpC orthologs of K. pneumoniae, E. aerogenes and E. cloacae have been reported 12,25,26. Another recent study reported th...

Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.