 Hi, my name is Karolina Majorek and I'm from Wadek Minor's lab at the University of Virginia in Charlottesville. I would like to encourage you to read our article, Double Trouble – Buffer Selection and Histide Presence might be responsible for non-reproducibility of biomedical experiments. It is the featured cover article of the October 2014 issue of Protein Science. In this article, we talk about the influence of polyhystidine affinity tags and buffer heaps on the outcome of protein functional and structural studies. Both of these are very important in the process of obtaining proteins for numerous experiments, but they have to be used with caution, as both might perturb the confirmation and activity of a protein. Reproducibility of experiments in other laboratories might be affected by these factors, even when changes between protocol seem to be insignificant. This might cause a ripple effect as subsequent studies are based on questionable data. Most in vitro biochemical and drug discovery experiments require large quantities of purified and active protein. Many technologies have been developed to increase the efficiency of protein production for in vitro experiments. Affinity tags provide a handle for easy purification of recombinant protein. Polyhystidine, or HIST tags, are especially popular since they are smaller than others, and thus should minimally perturb a protein structure and function. However, there is growing evidence to suggest that HIST tags may have significant effects on structural and activity measurements. Similarly, buffering agents are crucial for preserving a desired pH, but the correct choice of buffer agent is critical to avoid other unwanted chemical effects. Buffers have been shown to interfere with proteins by reacting with substrates or inhibiting enzymatic reactions. By structural and functional experiments, we observed the impact of both HIST tags and buffer molecules on three proteins in the GCN5-related N-acetyltransferase, or NAT, superfamily. Both the HIST tag and the buffer heapus influenced the confirmation and activity of these proteins. Our prior work showed that the pseudomonas aeruginosa protein PA4794 is a NAT acetyltransferase and determined the structure of the protein in complex with products of the reaction, coenzyme A, and acetylated peptide, shown here in pink. When PA4794 is expressed and crystallized in the presence of heapus, the heapus molecule binds in the substrate site in a position similar to that of peptide substrate. Heapus binding induces a conformational change between the apo form of PA4794 and the buffer bound form. The differences are located in the active site area and involve both main chain and side chains. These conformational changes are not observed in any of the other 18 structures of PA4794. If the heapus bound form was the only known structure, one might mistakenly conclude that this conformation was the apo form of the enzyme. When heapus was removed from purification and crystallization buffers, but the histag was not removed by proteolytic cleavage, the histag blocks the substrate binding site. The histag is not inserted as deeply into the active site as substrate or heaps, but occludes the site from outside. In kinetic experiments, the presence of the tag decreased the affinity of PA4794 for peptide substrate. We also analyzed the kinetic parameters of two other NAT proteins, SPEG, a spermedine-sperming acetyltransferase from Vibrio cholera and SA-COL-1063, an N-terminal protein acetyltransferase from Staphylococcus aureus. When both enzymes were tested with histag present, both exhibit decreased activity or affinity for both acetyl-coenzyme A and substrates, as compared to untagged enzyme. Even though we show just a few examples, the effects of buffers and histag are highly underestimated. In more than 18% of the hundreds of structures in the PDB containing heapus, the buffer molecule is bound in close proximity to the active site of proteins. Similarly, many structures of proteins contain evidence of histags bound in the proximity of active sites, but are either not discussed or are omitted altogether from the model, regardless of the crystallographic evidence of their presence. For example, in the structure 2QHA, the histag motif was not modeled, and it was claimed in the primary citation that the motif was not visible in the density map. However, there is clear density for the histag in the active site. When the glycerol and water molecules in this density was replaced with six histidine residues, a few cycles of refinement decreased the R and R-free of this structure by several percent. This shows how critical it is to adjust experimental protocols for each protein or family of proteins and investigate the influence of these factors to properly interpret protein activity and structure. Our results have shown the importance of these factors for members of the NAT superfamily, but may apply to interpreting results obtained for other proteins as well.