PubMed · 13726735
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R H MULLER. 1960-07-22. Automatic chemical analysis.. https://doi.org/10.1111/j.1749-6632.1960.tb23224.x
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The computation of minimum energy paths (MEPs) is an approach for gaining insight into protein conformational transitions that are too slow to be observed with unconstrained molecular dynamics simulations. MEPs have the advantage of providing the energy barrier of the rate-limiting step(s), allowing discrimination among different paths. Finding low-energy MEPs for complex transitions, such as those involving rearrangements of the backbone fold or repacking of buried side chains, has hitherto been unfeasible in a reliable, automated manner, the MEP often displaying unphysical behavior, such as the crossing of bonds. Here, this problem is addressed by combining a counterintuitive procedure for generating an initial guess of the path, in which all side chains are shrunk, with the conjugate peak refinement (CPR) method. The effectiveness of the approach is tested on the conformational switch in Ras p21. This conformational transition involves some partial unfolding and re-folding, a process for which a multitude of pathways are likely to exist and for which a single MEP does not provide a complete description. However, this transition requires some sterically demanding rearrangements, thus testing the ability of a method to find low-energy pathways free of structurally unphysical events. This is achieved by the present approach, which finds a path whose rate-limiting barrier is compatible with experiment. This demonstrates that the method can be used to compute plausible pathways for complex rearrangements in proteins in an automated manner that is unbiased by external driving constraints.
Establishing solubilization conditions for membrane-associated receptors is often a tedious empirical process. Here we describe a novel application of SPR biosensor technology to screen solubilization conditions automatically and to assess receptor activity directly. We focus on two chemokine receptors, CXCR4 and CCR5, which are important in HIV cell invasion. The autosampler in Biacore 3000 permitted whole cells expressing C-terminally tagged receptors to be automatically lysed under a given solubilization condition and the lysates to be injected over an antibody surface. The total amount of solubilized receptor could be quantitated from the antibody capture level, whereas the amount of active receptor could be quantitated using a subsequent injection of conformationally sensitive antibody or protein. Using this approach, we identified detergent/lipid/buffer combinations that enhanced and maintained receptor activity. We also used the biosensor to demonstrate CD4-dependent binding of gp120 to solubilized CCR5 and to develop affinity chromatography-based purification methods that increased receptor activity more than 300%. Together, these results illustrate the benefits of using the biosensor as a tool for isolating functional membrane receptors and for analyzing ligand/receptor interactions.
In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.