Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMPs activity. Relatively modest modifications to AMPs primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Gram-positive bacteria while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.
Photometric accuracy is determined by the difference between the measured absorbance and the established standard value. Most quantitation applications using UV-Vis would involve the measurement of the standards and samples of comparable concentrations in rapid succession on the same instrument. Photometric accuracy is critical for measurement; it compares the extinction coefficients between different instruments, for any photometric inaccuracy will lead to errors in quantitation. In Circular Dichroism (CD) measurements it is important and often a requirement to also monitor the absorbance of the sample. However, it is common to make separate measurements to determine the absorbance which is less accurate and time consuming. This study shows that the simultaneous measurement of ordinary UV/vis absorbance spectra from the photomultiplier high voltage output during a circular dichroism measurement is accurate and reliable. The photometric accuracy and linearity of various spectrometers is determined using standard solutions of potassium dichromate. The mathematical treatment of the signal representing the high voltage applied to the detector is presented here. An ordinary UV-vis spectrophotometer was used to measure the direct UV absorbance for comparison and to assign a reference value (Z-Value) to each CD spectrometer. The results proved to be accurate and reliable.
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