Although site-specific incorporation of artificial functionalities into proteins is an important tool in both basic and applied research, it can be a major challenge to protein chemists. Enzymatic protein modification is an attractive goal due to the inherent regio- and stereoselectivity of enzymes, yet their specificity remains a problem. As a result of the intrinsic reversibility of enzymatic reactions, proteinases can in principle catalyze ligation reactions. While this makes them attractive tools for site-specific protein bioconjugation, competing hydrolysis reactions limits their general use. Here we describe the design and application of a highly specific trypsin variant for the selective modification of N-terminal residues of diverse proteins with various reagents. The modification proceeds quantitatively under native (aqueous) conditions. We show that the variant has a disordered zymogen-like activation domain, effectively suppressing the hydrolysis reaction, which is converted to an active conformation in the presence of appropriate substrates.

SummaryPhage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ 54 enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ 54 -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiledcoil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains.

Among the strategies that employ genetic engineering to stabilize proteins, the introduction of disulfide bonds has proven to be a very potential approach. As, however, the replacement of amino acid residues by cysteines and the subsequent formation of the covalent bond can result in a severe deformation of the parental protein structure, the stabilization effect is strongly context dependent. Alternatively, the introduction of charged amino acid residues at the surface, which may result in the formation of extra ionic interactions or hydrogen bonds, provide propitious means for protein stabilization. The generation of an extra disulfide bond between residues 4 and 118 in ribonuclease A had resulted in a stabilization by 6 °C or 7 kJ mol−1, which was mainly caused by a deceleration of the unfolding reaction [Pecher, P. & Arnold, U. (2009) Biophys Chem, 141, 21–28]. Here, Asp83 was replaced by Glu resulting in a comparable stabilization. Moreover, combination of both mutations led to an additive effect and the resulting ribonuclease A variant (Tm ∼ 76 °C, ΔG°∼ 53 kJ mol−1) is the most stable ribonuclease A variant described so far. The analysis of the crystal structure of A4C/D83E/V118C‐ribonuclease A reveals the formation of a salt bridge between the γ‐carboxyl group of Glu83 and the ε‐amino group of Lys104. Database Ribonuclease (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/1/27/5.html) Structured digital abstract  http://www.uniprot.org/uniprot/P61823 and http://www.uniprot.org/uniprot/P61823 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8375221)

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Artifizielle Funktionalisierungen von Proteinen stellen sowohl in der angewandten als auch in der Grundlagenforschung ein wichtiges Werkzeug dar und erweisen sich als eine Herausforderung für Proteinchemiker. Aufgrund ihrer nativen Regio-und Stereoselektivität bieten enzymatische Proteinmodifizierungen einen attraktiven Ansatz, jedoch ist eine universelle Anwendung durch deren hohe Spezifität beschränkt. Aufgrund der intrinsischen Reversibilität enzymatischer Reaktionen sind Proteinasen prinzipiell in der Lage auch Ligationen zu katalysieren. Dies macht sie zu einem interessanten Werkzeug für spezifische Proteinkonjugationen. Wir berichten über die Entwicklung einer hochspezifischen Trypsinvariante für die selektive N-terminale Modifizierung von Proteinen. Die Reaktion verläuft mit quantitativen Produktausbeuten unter nativen Bedingungen. Wir zeigen, dass die Variante eine ungeordnete Zymogen-ähnliche Aktivierungsdomäne aufweist, die in Gegenwart geeigneter Substrate in die aktive Konformation überführt wird.