A post-translational modi®cation affecting the translation termination rate was identi®ed in the universally conserved GGQ sequence of release factor 2 (RF2) from Escherichia coli, which is thought to mimic the CCA end of the tRNA molecule. It was shown by mass spectrometry and Edman degradation that glutamine in position 252 is N 5 -methylated. Overexpression of RF2 yields protein lacking the methylation. RF2 from E.coli K12 is unique in having Thr246 near the GGQ motif, where all other sequenced bacterial class 1 RFs have alanine or serine. Sequencing the prfB gene from E.coli B and MRE600 strains showed that residue 246 is coded as alanine, in contrast to K12 RF2. Thr246 decreases RF2-dependent termination ef®ciency compared with Ala246, especially for short peptidyltRNAs. Methylation of Gln252 increases the termination ef®ciency of RF2, irrespective of the identity of the amino acid in position 246. We propose that the previously observed lethal effect of overproducing E.coli K12 RF2 arises through accumulating the defects due to lack of Gln252 methylation and Thr246 in place of alanine.

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z SPA-1 affibody has been selected from a phagedisplayed library as a binder to protein A. Z SPA-1 also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z SPA-1 affibody in its uncomplexed state and determined the solution structure of a Z:Z SPA-1 protein-protein complex. Uncomplexed Z SPA-1 behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z SPA-1. We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z SPA-1 and Ig Fc, respectively, suggesting that the Z SPA-1 affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.protein engineering ͉ protein-protein interactions ͉ molecular recognition ͉ NMR spectroscopy ͉ induced fit T here is an interest in generating novel classes of binding proteins that can be used as an alternative to immunoglobulins in various biochemical assays and biotechnological applications. To this end, carefully chosen protein domains can be used as framework structures for combinatorial protein engineering. Affibodies constitute a class of engineered binding proteins for which the three-helix bundle Z domain is used as a scaffold. The 58-aa residue Z domain is derived from one of five homologous domains (the B domain) in Staphylococcus aureus protein A (SPA). SPA binds strongly to the Fc region of immunoglobulins, and Z was originally developed as a stabilized gene fusion partner for affinity purification of recombinant proteins by using IgG-containing resins (1). The structure of a complex between the B domain of SPA and an Fc fragment shows that the binding surface consists of residues that are exposed on helices 1 and 2, whereas helix 3 is not directly involved in binding (2). Affibodies are selected from combinatorial libraries in which typically 13 residues at the Fc-binding surface of helices 1 and 2 are randomized. Specific binders to target proteins are then identified by biopanning the phage-displayed library against desired targets (3). Several Z-based affibodies with specific proteinbinding properties have in this way been developed and used as affinity tools in a number of applications (4-7).Structural studies of engineered protein-binding domains and their complexes are of interest for methods development in biotechnology as well as for basic studies of protein-protein interactions and the mechanisms of biomolecular recognition. Here we describe the (solution) structural and biophysic...

SummaryDicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. In addition to revealing the dual RNA and protein binding properties of Dicer C-terminus, our results may provide a link between miRNA-mediated regulation of gene expression and inflammation.

We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA)(-)(1) affibody and it's Z domain binding partner as a model. Free Z(SPA)(-)(1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA)(-)(1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA)(-)(1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA)(-)(1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA)(-)(1) complex is associated with a large change in conformational entropy that opposes binding.

Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z SPA-1 affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z SPA-1 complex and noted that Z SPA-1 in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z SPA-1 and four Z SPA-1 mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z SPA-1 was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z SPA-1 is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z SPA-1 with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.Keywords: protein engineering; affibody; protein stability; osmolyte; NMR spectroscopy Several biochemical and biotechnological applications rely on proteins with specific recognition properties. Antibodies have for a long time been, and are still in most cases, the obvious choice, but recent combinatorial approaches have resulted in several novel kinds of affinity proteins (Nygren and Uhlén 1997). One class of such specific binding proteins is called affibodies. These are based on the 58-residue scaffold of the Z domain-a one-domain staphylococcal protein A (SPA) analog (Nilsson et al. 1987). The Z domain possesses several properties suitable for an artificial binding protein: it is small, soluble, stable, and easily produced with common expression systems. Affibodies are selected from phagemid libraries of Z-domain constructs in which 13 solvent accessible residues are subjected to combinatorial mutagenesis (Nord et al. 1997). The residues to be randomized were chosen based on the interaction surface for the Zdomain interaction with the Fc fragment from IgG (Deisenhofer 1981). Specific binders are then obtained by phagedisplay screening against the desired target protein. The methodology has resulted in the identification of several affibodies with high specificity for the applied panning target (Hansson et al. 1999;Nord et al. 2001;Rönnmark et al. 2002;Sandström et al. 2003 N heteronuclear single quantum correlation; ANS, 8-anilino-1-naphtal...

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