A new Differential Scanning Calorimeter for the multi-user Molecular Biophysics Research Centre

点击翻译按钮获取中文摘要

Differential Scanning Calorimetry (DSC) is an important biophysical technique that can be used to determine the stability of proteins or the stability of lipid layers as a function of temperature. By gradually increasing the temperature in a specific ‘thermo-controlled’ compartment one can obtain an accurate estimate of the denaturation temperature of a protein. The denaturation temperature of the protein - the point where it looses its 3D structure - typically increases when ligands bind to the protein. The extent of this increase is directly related to the strength of the interaction. All commercially available DSC instruments can carry out these measurements in an automated fashion. Hence DSC has become an important method to compare the binding of various ligands, such as enzyme substrates, co-factors or co-enzymes, drugs, drug leads, metal ions or regulatory compounds to purified proteins. In the pharmaceutical industry DSC is being used extensively to study the stability of protein therapeutics and to screen for optimal formulation conditions. The main advantage of DSC is that it is a completely label-free measurement: the parameter measured is the heat that is absorbed (or produced) when proteins unfold and refold. Lipid phase transitions can also be followed directly by DSC measurements. In recent years much work has focused on studies of phospholipids, the major constituents of biological membranes. Distinct phospholipid bilayers and multilayers have characteristic transitions in their phase behaviour that occur at specific temperatures depending on the length of their fatty acid chains and the chemical nature of their phospholipid headgroups. When transmembrane proteins or perturbing agents, such as antimicrobial or anticancer peptides, are binding to these membrane mimetics, the phase behavior changes, often creating distinct patches of peptides that are surrounded by selected lipids, which then display different transition temperatures. Hence in this case the label-free DSC measurements help to characterize the interplay between the lipids and the other components of biological membranes. Why do we apply now for a DSC instrument? In 2001 we were fortunate that we could purchase our first Microcal VP-DSC instrument. The instrument has been in continued use and it has served us well over the years; the results obtained have been included in numerous published manuscripts. The instrument is incorporated in the multiuser Biophysics Research Centre, which has users from all across our campus. The original DSC instrument has recently developed a leak and the manufacturer has quoted us a steep price to have the instrument repaired. Given the age of the instrument (which includes an outdated obsolete computer system) the repair cost seems exorbitant. We have therefore decided that the time has come to replace it with a new device that can provide us with reliable data for the next 15 years.