Lars Baltzer

Structure and dynamics of a designed helix-loop-helix dimer in dilute aqueous trifluoroethanol solution. A strategy for NMR spectroscopic structure determination of molten globules in the rational design of native-like proteins

BACKGROUND The overwhelming majority of engineered amino acid sequences designed to fold into well defined tertiary structures show the hallmarks of molten globules. Although imperfectly folded, the structures of these polypeptides are of considerable interest in assessing the predictive power of design strategies and in understanding the structural basis for the formation of proteins with native-like properties. This paper describes a strategy for the structural characterization of molten globules by NMR spectroscopy applied to the study of SA-42, a polypeptide with 42 amino acids that folds into a hairpin helix-loop-helix dimer. RESULTS The 1H NMR spectrum of SA-42 was assigned in several mixtures of water and trifluoroethanol (TFE) (0-30 vol%) and small amounts of TFE were shown to have a significant effect on the spectrum. The secondary and supersecondary structures of SA-42 were determined. In aqueous solution a helix-loop-helix dimer is formed, but in 30 vol% of TFE the population of hairpin dimers are negligible and SA-42 is monomeric, folding into two non-interacting helical segments. In solutions containing less than 3 vol% of TFE the structure is very similar to that in water and the structural information may be used to develop the motif in aqueous solution. Less well ordered amino acid residue sidechains in the hydrophobic core were identified. Helix distortion in the tetrahelix bundle was found to be small. CONCLUSIONS Detailed information about molten globule structures in aqueous solution can be obtained from NMR spectroscopy if the spectra are assigned in dilute TFE solution. On the basis of the NMR spectroscopic analysis, the solution structure of SA-42 was found to be close to the designed one. A route for developing native-like properties in SA-42 is suggested based on the identification by NMR spectroscopy of some less well ordered amino acid sidechains in the hydrophobic core and on the observed structural rigidity of the two helices.

Functionalization and Properties of Designed Folded Polypeptides

Designed, folded and functionalized polypeptides and proteins constitute an enormous pool of new shapes, new functions and new materials. By taking advantage in the chemical laboratory of the principles of protein folding used by nature, strategies have so far been developed for the engineering of new catalysts, metalloproteins, heme proteins, glycoproteins, receptors and mimics of the components of the immune system. Catalysts have been developed that catalyze reactions not performed by nature and uncommon folded polypeptide motifs have been engineered and structurally characterized. The search for and exploitation of the tremendous number of proteins yet to be discovered has thus begun. Understanding of the protein folding problem has now reached a level where the design of peptides that approach a hundred residues in size is feasible, although not trivial, and clearly sequence dependent. The most frequently designed motif is the four-helix bundle, but recently monomeric triple-stranded β-sheet structures have also been reported as well as a ββα-motif, helical coiled coils and triple helices. Template-assembled polypeptides as well as linear sequences have been shown to fold into designed solution structures and these and other motifs are now key targets for functionalization. This review describes the principles and strategies used in the design of these motifs, as well as their structural characterization. Strategies for functionalization using both the naturally occurring amino acids and post-synthetic incorporation of non-natural functionality will be described, as well as the level of function that has been achieved by rational design.

De novo designed polypeptide catalysts with adopted folded structures

Designed polypeptide catalysts have been shown to catalyze hydrolysis and transesterification reactions of p-nitrophenyl esters by a mechanism that includes the nucleophilic attack by an unprotonated histidine and general-acid catalysis by a flanking protonated histidine. The catalysis is cooperative and exhibits rate enhancements of three orders of magnitude over that of the 4-methylimidazole catalyzed reaction. Substrate recognition by residues introduced in the adjoining helix was demonstrated for the negatively charged substrate mono-p-nitrophenyl fumarate. The results have been compared to those obtained for other designed polypeptide catalysts with similar efficiency, and it was concluded that the hallmarks of naturally occurring biocatalysts have now been demonstrated in polypeptide catalyzed reactions, although with considerably less efficiency than native enzymes. It was found that so far the most severe limitation of folded polypeptide catalysts is the efficiency obtained in the bond-making and bond-breaking steps, whereas the binding of substrates, even on the surface of helical structures in aqueous solution, is of comparable strength to that which occurs in nature.

The pH-dependent tertiary structure of a designed helix–loop–helix dimer

BACKGROUND De novo designed helix-loop-helix motifs can fold into well-defined tertiary structures if residues or groups of residues are incorporated at the helix-helix boundary to form helix-recognition sites that restrict the conformational degrees of freedom of the helical segments. Understanding the relationship between structure and function of conformational constraints therefore forms the basis for the engineering of non-natural proteins. This paper describes the design of an interhelical HisH+-Asp- hydrogen-bonded ion pair and the conformational stability of the folded helix-loop-helix motif. RESULTS GTD-C, a polypeptide with 43 amino acid residues, has been designed to fold into a hairpin helix-loop-helix motif that can dimerise to form a four-helix bundle. The folded motif is in slow conformational exchange on the NMR timescale and has a well-dispersed 1H NMR spectrum, a narrow temperature interval for thermal denaturation and a near-UV CD spectrum with some fine structure. The conformational stability is pH dependent with an optimum that corresponds to the pH for maximum formation of a hydrogen-bonded ion pair between HisH17+ in helix I and Asp27- in helix II. CONCLUSIONS The formation of an interhelical salt bridge is strongly suggested by the pH dependence of a number of spectroscopic probes to generate a well-defined tertiary structure in a designed helix-loop-helix motif. The thermodynamic stability of the folded motif is not increased by the formation of the salt bridge, but neighbouring conformations are destabilised. The use of this novel design principle in combination with hydrophobic interactions that provide sufficient binding energy in the folded structure should be of general use in de novo design of native-like proteins.

Covalent control of polypeptide folding. Induction of helix-loop-helix motifs by bridging

A de novo designed helix-loop-helix polypeptide motif is induced by introduction of an interhelical carbon bridge by a site selective reaction. The proposed structure of the bridged polypeptide is based on CD spectroscopy and previous structural studies of polypeptides with similar amino acid sequences. The polypeptide used for the bridging experiments is PE42Dcap, a de novo designed polypeptide with 42 amino acid residues that has been designed to fold into a helix-loop-helix motif and to dimerise to form four-helix bundles in aqueous solution. PE42Dcap is engineered to perform acyl transfer reactions with bifunctional esters yielding an interhelical, covalent bridge between two lysine residues. The active residues in PE42Dcap are His7, His11 and Lys15 in helix I and His26, His30 and Lys34 in helix II. Reaction of PE42Dcap with disuccinimidyl glutarate at pH 4.1 in sodium acetate buffer and 5 vol% 2,2,2-trifluoroethanol gave the pure bridged polypeptide DSG-PE42Dcap in an isolated yield of 35%. CD spectroscopy on the precursor polypeptide PE42Dcap gave the absolute mean residue ellipticity −4800 deg cm2 dmol−1 at 222 nm in aqueous solution at pH 5.1. This shows that the helical content in the polypeptide is low. The introduction of the interhelical carbon bridge increased the absolute mean residue ellipticity to −20100 deg cm2 dmol−1, showing that the bridge has dramatically increased the helical content and that DSG-PE42Dcap is mainly present as helix-loop-helix motifs. The mean residue ellipticity of DSG-PE42Dcap shows concentration dependence that indicates that this bridged polypeptide is mainly monomeric at micromolar concentrations and dimeric above 100 μM. p

Catalysis of pyridoxal phosphate mediated transamination

The design of folded polypeptide catalysts is a new way of studying enzyme catalysis and opens up new routes to biocatalysts that can be used in the chemical laboratory and in the large-scale production of organic compunds using fermentation technology. The design of folded polypeptide catalysts is based on the introduction of reactive sites into protein templates or scaffolds that have enough inherent binding energy to sustain the sequence modifications needed to establish the relationship between structure and function. We have developed 42-residue helix-loop-helix motifs that dimerize in solution to form four-helix bundles, and introduced reactive sites for the catalysis of acyl-transfer reactions [1-3] and more recently of decarboxylation reactions. Here we wish to report on the catalysis of a considerably more complex reaction, the transamination of amino acids to form the corresponding acid, in order to develop by rational design a catalytic system capable of multiple turnovers in the synthesis of artificial amino acids. The catalyst depends on the cofactor pyridoxal phosphate, used by nature in the biosynthesis of amino acids.

HisH+-His reactive sites in catalytic four-helix bundle catalysts

Helix-loop-helix motifs with 42 amino acid residues that dimerize to four-helix bundles have previously been designed to catalyse the hydrolysis of activated esters. The catalysts exhibit rate enhancements of more than three orders of magnitude, chiral discrimination and saturation kinetics [1,2]. The suggested mechanism includes cooperative nucleophilic and general acid catalysis by HisH+-His pairs.We have now expanded the concept of His catalysis to new reactive site structures and determined conditions under which the generalacid catalysis becomes important.