The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

Telomeric C-rich strands can form a noncanonical intercalated DNA structure known as an i-motif. We have studied the interactions of the cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) with the i-motif forms of several oligonucleotides containing telomeric sequences. TMPyP4 was found to promote the formation of the i-motif DNA structure. On the basis of (1)H NMR studies, we have created a model of the i-motif-TMPyP4 complex that is consistent with all the available experimental data. Two-dimensional NOESY data prompted us to conclude that TMPyP4 binds specifically to the edge of the intercalated DNA core by a nonintercalative mechanism. Since we have shown that TMPyP4 binds to and stabilizes the G-quadruplex form of the complementary G-rich telomeric strand, this study raises the intriguing possibility that TMPyP4 can trigger the formation of unusual DNA structures in both strands of the telomeres, which may in turn explain the recently documented biological effects of TMPyP4 in cancer cells.

The time course of refolding of both pig muscle and yeast 3-phosphoglycerate kinase (molecular masses about 47 kDa), as well as their proteolytic C-terminal fragments (30 and 33 kDa, respectively) has been investigated. Very similar refolding kinetics (with half-time between 80-120 s, at 20°C) were observed by fluorescence and ultraviolet absorbance spectroscopy, as well as by activity measurements, for the intact enzyme from both sources. This time course appears not to depend on the time the protein spends in the unfolded state, i.e. it is certainly not controlled by proline isomerization. Furthermore, after removal of a large N-terminal part (molecular mass of about 18 kDa for pig muscle enzyme or 13 kDa for yeast enzyme) of the molecule by proteolysis, refolding of the remaining C-terminal fragment of both proteins follows kinetics virtually indistinguishable from those of the intact protein molecule.The refolding of simple globular proteins occurs on a wide range of time scale, from the subsecond region [l, 21 up to minutes or hours [2-61. In some cases this process can be resolved into kinetically distinguishable steps (e. g. [7, 81). However, the nature of the folding intermediates and the ratedetermining steps could rarely be identified (cf. reviews [9, 101). If the protein contains no disulfide bridges, the only reason known for slow refolding is the isomerization around the proline imide peptide bond [l 1 -151 which can be accelerated by a specific enzyme, proline cis-trans-isomerase [13, 151. Apart from this, other types of unknown slow conformational processes might also be operative in the folding pathway. For example, kinetic investigation of horse muscle 3-phosphoglycerate kinase refolding revealed a slow phase which was not attributable to proline isomerization [16]. This fact requires special attention, since it may reflect the presence of an unknown rate-limiting factor in protein refolding. It is possible that the slow refolding of 3-phosphoglycerate kinase is somehow associated with the two-domain structural nature of the molecule as the correct domain assembly can be the rate-limiting step. A useful approach to clarify this point is the separation of the C-terminal and N-terminal parts of the molecule and the study of their refolding separately [24]. Various efforts have succeeded in producing large fragments (or domains) of the enzyme molecule either by analytical 1251 and proteolytic [26, 271 cleavage or by genetic engineering [28, 291. Renaturation experiments with the fragments supported their ability to fold independently both for horse muscle [26] and yeast [25, 301 3-phosphoglycerate kinases. Nevertheless, no kinetic experiment has yet been performed to study the refolding of the isolated fragments of this enzyme. We first showed the possibility of reactivation of pig muscle 3-phosphoglycerate kinase from its isolated and unfolded proteolytic fragments [31]. To understand further the rate-limiting step of the protein refolding, we investigated the refolding of one isolated C-termin...

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The de novo protein albebetin has been designed recently to form a predetermined tertiary fold that has not yet been observed in natural proteins. An eight amino acid fragment (131-138) of human interferon alpha(2) carrying the blast-transforming activity of the protein was attached to the N-terminus of albebetin next to its initiatory methionine residue. The gene of chimeric protein was expressed in a wheat germ cell-free translation system and synthesized protein was tested for its compactness and stability. Its ability for receptor binding was also studied. We have shown that albebetin with attached octapeptide is practically as compact as natural proteins of corresponding molecular weight and possesses high stability toward the urea-induced unfolding. It binds murine thymocyte receptor at a high affinity and activates the thymocyte blast transformation efficiently at a concentration of 10(-11) M.