Although some degradants can potentially influence the stability of the protein (as discerned from spiking studies), degradation of polysorbates did not impact the stability of the different proteins tested in pharmaceutically relevant temperature and storage conditions.

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Peptide-preservative interactions not only affect peptide self-interactions, but also antimicrobial efficiency of the preservatives and are thus of significant relevance. Adsorption of preservatives on oligomeric states of peptides is proposed as a mechanism to explain this reduced antimicrobial efficacy.

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The crystal structure of the thermostable indoleglycerol-phosphate synthase from Thermotoga maritima (tIGPS) was determined at 2.5 Å resolution. It was compared with the structures of the thermostable sIGPS from Sulfolobus solfataricus and of the thermolabile eIGPS from Escherichia coli. The main chains of the three (␤␣) 8 ؊barrel proteins superimpose closely, and the packing of side chains in the ␤-barrel cores, as well as the architecture of surface loops, is very similar. Both thermostable proteins have, however, 17 strong salt bridges, compared with only 10 in eIGPS. The number of additional salt bridges in tIGPS and sIGPS correlates well with their reduced rate of irreversible thermal inactivation at 90°C. Only 3 of 17 salt bridges in tIGPS and sIGPS are topologically conserved. The major difference between the two proteins is the preference for interhelical salt bridges in sIGPS and intrahelical ones in tIGPS. The different implementation of salt bridges in the closely related proteins suggests that the stabilizing effect of salt bridges depends rather on the sum of their individual contributions than on their location. This observation is consistent with a protein unfolding mechanism where the simultaneous breakdown of all salt bridges is the rate-determining step.It is important to understand how thermophilic organisms stabilize their proteins (1-3). Further insights into the structural features essential for protein stability will improve our understanding of how protein thermostability may have been lost or gained during evolution, and how proteins from mesophilic organisms might be stabilized for industrial applications (4).Comprehensive comparisons of structural features of protein families from mesophiles and thermophiles (5, 6) have confirmed earlier observations that the majority of thermostable proteins has an increased number of salt bridges, as compared with their thermolabile counterparts. The picture of salt bridges generally strengthening thermostable proteins has, however, been modified by recent work. Computational studies (7), as well as tests of specific stabilizing interactions in small proteins conducted by replacing specific residues (8 -11), have shown that the decisive feature is rather the optimal placement of charged residues on the protein surface.We are interested in how the (␤␣) 8 Ϫbarrel fold (12), which is frequently observed in enzymes with quite different functions (13), is stabilized in thermophilic organisms. We have previously studied the (␤␣) 8 -barrel enzymes phosphoribosyl-anthranilate isomerase (PRAI) 1 and indoleglycerol-phosphate synthase (IGPS), which catalyze two consecutive steps in the pathway of tryptophan biosynthesis. In Escherichia coli (14), IGPS and PRAI are the covalently linked domains of the bifunctional enzyme eIGPS-ePRAI. The genetically separated ePRAI and eIGPS domains are fully active monomers (15), but their covalent linkage in the parental protein is advantageous, because it stabilizes the particularly labile eIGPS domain.Previous comparisons of...