Francisco J. Blanco

The order of secondary structure elements does not determine the structure of a protein but does affect its folding kinetics.

We have analyzed the structure, stability and folding kinetics of circularly permuted forms of alpha-spectrin SH3 domain. All the possible permutations involving the disruption of the covalent linkage between two beta-strands forming a beta-hairpin have been done. The different proteins constructed here fold to a native conformation similar to that of wild-type protein, as demonstrated by nuclear magnetic resonance and circular dichroism. Although all the mutants have similar stabilities (they are 1 to 2 kcal mol-1 less stable than the wild-type) their rate constants for folding and unfolding are quite different. Protein engineering, in combination with kinetics indicates that the folding pathway has been changed in the circularly permuted proteins. We conclude that neither the order of secondary structure elements, nor the preservation of any of the beta-hairpins present in this domain, is crucial for the ability of the polypeptide to fold, but they influence the folding and unfolding kinetics and could determine its folding pathway.

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265u2009bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein-DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo.

Solution structure and NMR characterization of the binding to methylated histone tails of the plant homeodomain finger of the tumour suppressor ING4

Plant homeodomain (PHD) fingers are frequently present in proteins involved in chromatin remodelling, and some of them bind to histones. The family of proteins inhibitors of growth (ING) contains a PHD finger that bind to histone‐3 trimethylated at lysine 4, and those of ING1 and ING2 also act as nuclear phosphoinositide receptors. We have determined the structure of ING4 PHD, and characterised its binding to phosphoinositides and histone methylated tails. In contrast to ING2, ING4 is not a phosphoinositide receptor and binds with similar affinity to the different methylation states of histone‐3 at lysine 4.

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: comparison of unrefined and refined structure sets with the crystal structure.

The assignment of the 1H and 15Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain α-spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.

Elongation of the BH8 β-hairpin peptide: Electrostatic interactions in β-hairpin formation and stability

An elongated version of the de novo designed β‐hairpin peptide, BH8, has allowed us to gain insight into the role of electrostatic interactions in β‐hairpin stability. A Lys–Glu electrostatic pair has been introduced by adding a residue at the beginning and at the end of the N‐terminal and C‐terminal strands, respectively, of the β‐hairpin structure, in both orientations. The two resulting peptides and controls having Ala residues at these positions and different combinations of Ala with Lys, or Glu residues, have been analyzed by nuclear magnetic resonance (NMR), under different pH and ionic strength conditions. All of the NMR parameters, in particular the conformational shift analysis of Cα protons and the coupling constants, 3JHNα, correlate well and the population estimates are in reasonable agreement among the different methods used. In the most structured peptides, we find an extension of the β‐hairpin structure comprising the two extra residues. Analysis of the pH and salt dependence shows that ionic pairs contribute to β‐hairpin stability. The interaction is electrostatic in nature and can be screened by salt. There is also an important salt‐independent contribution of negatively charged groups to the stability of this family of β‐hairpin peptides.

Role of a nonnative interaction in the folding of the protein G B1 domain as inferred from the conformational analysis of the α-helix fragment

BACKGROUNDnThe role of local interactions in protein folding and stability can be investigated by the conformational analysis of protein fragments. The hydrophobic staple and Schellman motifs have been described at the N and C terminus, respectively, of protein alpha-helices. These motifs are characterized by an interaction between two hydrophobic residues, one outside the helix and one within the helix, and their importance for helix stability has been analyzed in model peptides. In the alpha-helix of the protein G B1 domain, only the Schellman motif is formed--the hydrophobic staple motif is absent despite the favourable sequence pattern. We have experimentally analyzed the solution conformation of the 19-41 fragment of protein G. This peptide comprises the helical residues and contains both the hydrophobic staple and Schellman motif sequences.nnnRESULTSnIn the isolated peptide in water, the hydrophobic staple motif is formed and stabilizes the helical structure as compared with a shorter peptide lacking it, but the Schellman motif is not formed. In 30% aqueous TFE, the helix is more stable than in pure water and both motifs are formed.nnnCONCLUSIONSnThe results suggest that the importance of each motif for the folding and stability of protein G is different. The nonnative hydrophobic staple interaction can help to nucleate the helix at the beginning of folding but has later to be disrupted. The Schellman motif, while not providing enough energy for substantial helix stabilization in the unfolded state, could be important for determining the local fold of the sequence in the context of the rest of the protein.

Infrared evidence of a β-hairpin peptide structure in solution

The IR spectrum of an 16‐amino acid peptide corresponding, according to NMR studies, to a β‐hairpin has been analysed. Two characteristic features distinguish its spectrum from that of an antiparallel β‐sheet: the low‐frequency band that in a β‐sheet structure is located at 2 ∼ 1632 cm−1 appears here at 2∼ 1620 cm−1, and the high‐frequency component does not undergo the isotopic shift typical of β‐sheet from 1690 to 1675 cm−1 when transferred to D2O. The infrared characteristics associated with β‐hairpins have been described so far in two proteins, in one of which, whose three‐dimensional structure is known from X‐ray diffraction, a β‐hairpin has actually been detected.