Synthetic biology of proteins: tuning GFPs folding and stability with fluoroproline.

BACKGROUND: Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C(gamma)-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. C(gamma)-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides. METHODOLOGY/PRINCIPAL FINDINGS: In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the C(gamma)-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines. CONCLUSIONS/SIGNIFICANCE: We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the C(gamma)-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms.

Chymotryptic specificity determinants in the 1.0 A structure of the zinc-inhibited human tissue kallikrein 7.

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70-80 loop and the unique S1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by Zn(2+) and Cu(2+) at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at His(99) close to the catalytic triad accounts for the noncompetitive Zn(2+) inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition.

Open and closed structures of the UDP-glucose pyrophosphorylase from Leishmania major.

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel beta-helix (LbetaH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.

Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination.

Global replacements of tyrosine by 2- and 3-fluorotyrosine in "enhanced green" and "enhanced yellow" mutants of Aequorea victoria green fluorescent proteins (avGFPs) provided protein variants with novel biophysical properties. While crystallographic and modeled structures of these proteins are indistinguishable from those of their native counterparts (i.e., they are perfectly isomorphous), there are considerable differences in their spectroscopic properties. The fluorine being an integral part of the avGFP chromophore induces changes in the titration curves, variations in the intensity of the absorbance and fluorescence, and spectral shifts in the emission maxima. Furthermore, targeted fluorination in close proximity to the fluorinated chromophore yielded additional variants with considerably enhanced spectral changes. These unique spectral properties are intrinsic features of the fluorinated avGFPs, in the context of the rigid chromophore-microenvironment interactions. The availability of the isomorpohous crystal structures of fluorinated avGFPs allowed mapping of novel, unusual interaction distances created by the presence of fluorine atoms. In addition, fluorine atoms in the ortho position of the chromophore tyrosyl moiety exhibit a single conformation, while in the meta position two conformer states were observed in the crystalline state. Such global replacements in chromophores of avGFPs and similar proteins result in "atomic mutations" (i.e., H --> F replacements) in the structures, offering unprecedented opportunities to understand and manipulate the relationships between protein structure and spectroscopic properties.

A Salmonella fim homologue in Citrobacter freundii mediates invasion in vitro and crossing of the blood-brain barrier in the rat pup model.

From the invasive Citrobacter freundii strain 3009, an invasion determinant was cloned, sequenced, and expressed. Sequence analysis of the determinant showed high homology with the fim determinant from Salmonella enterica serovar Typhimurium. The genes of the invasion determinant directed invasion of recombinant Escherichia coli K-12 strains into human epithelial cell lines of the bladder and gut as well as mannose-sensitive yeast agglutination and were termed fim(Cf) genes. Expression of the Fim(Cf) proteins was shown by (35)S labeling and/or Western blotting. In the infant rat model of experimental hematogenous meningitis, C. freundii strain 3009 and the in vitro invasive recombinant E. coli K-12 strain harboring the fim(Cf) determinant reached the cerebrospinal fluid, in contrast to the case for the control strain. The fim determinant was also necessary for efficient in vitro invasion by C. freundii, because a deletion mutant was strongly reduced in its invasion efficiency. The mutation could be complemented in trans by the corresponding genes. Invasion by C. freundii could be blocked only by d-mannose, GlcNAc, and chitin hydrolysate and not by other carbohydrates tested. In contrast, yeast agglutination was not affected by GlcNAc or chitin hydrolysate. This finding indicated mannose residues to be essential for both yeast agglutination and invasion, whereas GlcNAc (oligomer) residues of host cells are involved exclusively in invasion. These results showed the fim determinant of C. freundii to be responsible for d-mannose- and GlcNAc-dependent in vitro invasion without being assembled into pili and for crossing of the blood-brain barrier in the infant rat model.