Published: February 22, 2011 r2011 American Chemical Society 2360 dx.doi.org/10.1021/jp107623n | J. Phys. Chem. B 2011, 115, 2360–2370 ARTICLE pubs.acs.org/JPCB Structural Conversion of Intramolecular and Intermolecular G-Quadruplexes of bcl2mid: The Effect of Potassium Concentration and Ion Exchange Chang-Ting Lin, †,‡ Ting-Yuan Tseng, †,‡ Zi-Fu Wang, †,§ and Ta-Chau Chang* ,†,‡,§ † Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Taipei 10617, Taiwan, Republic of China ‡ Institute of Biophotonics, National Yang-Ming University, Taipei 11221, Taiwan, Republic of China § Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan, Republic of China ’ INTRODUCTION A large number of potential G-quadruplex-forming sequences are found in the human genome. 1-3 Among them, the 3 0 - overhang G-rich single strand of human telomeres can form G-quadruplex (G4) structures under physiological conditions both in vitro 4,5 and In Vivo in metaphase chromosomes. 6,7 The folding of telomeric DNA into G4 structures is important in inhibiting the activity of telomerase; 8,9 thus, such a structure might be a potential target for the therapeutic intervention of cancer. 10-12 In this case, it would be important to know telomeric G4 structure for drug design. However, the G-rich sequences can adopt various G4 structures and can possibly coexist in mixtures. For example, NMR analysis showed that the human telomeric sequence, AG 3 (T 2 AG 3 ) 3 (HT22), forms an antiparallel basket G4 structure in a Na þ solution, 4 while X-ray crystallography showed that HT22 forms a parallel propeller G4 structure in the presence of K þ . 5 On the other hand, the precise G4 structures of HT22 and (T 2 AG 3 ) 4 (HT24) in K þ solution still remain undetermined, 13-16 which is due to the coexistence of two different intramolecular G4 structures that cause problems for structural analysis. 17,18 In addition, a spectral conversion of HT24 from the “sodium form” to the “potassium form” has been observed upon K þ titration. 18-23 Two possible mechanisms involving structural changes between different types of structures via unfolding 18-20 and structural changes within a single con- formational state without unfolding 21-23 were proposed for this spectral conversion. Investigation of structural diversity among various G4 structures and verification of the proposed structural conversion are essential for developing new anticancer drugs and for exploring their potential biological roles. 24,25 We propose the G-rich sequence with various G4 structures that can be easily separated and identified may be useful in verifying the proposed mechanisms for structural conversion. Since the intramolecular and intermolecular conformations can be easily distinguished by gel assays, a G-rich sequence that can adopt intra- and intermolecular G4 structures under different conditions is a good candidate to study when investigating the mechanisms of structural conversion. Gabelica et al. 26 found that the formation of intermolecular G4 assemblies is favored by short loops in the K þ solution. In addition to human telomeric G-quadruplexes, a number of nontelomeric G4 structures have been identified in human gene promoters such as the c-myc oncogene, 27-30 the bcl-2 gene, 31-33 the VEGF gene, 34 the KRAS gene, 35 and the c-kit oncogene. 36 Such promoters that can form G4 structures may play a critical role in gene transcription and regulation. 37 Hurley et al. 28 have examined the G-quadruplexes of AG 3 TG 4 AG 3 TG 4 (c-myc18) and TG 4 AG 3 TG 4 AG 3 TG 4 AAG 2 (c-myc27) in the c-myc gene Received: August 12, 2010 Revised: January 13, 2011 ABSTRACT: The gel assay, circular dichroism, and differential scanning calorimetry results all demonstrate that a major monomer component of bcl2mid exists at low [K þ ] and an additional dimer component appears at high [K þ ]. This implies that bcl2mid is a good candidate for elucidating the mechanisms of structural conversion between different G-quadruplexes. We further discovered that the conversion between the monomer and dimer forms of bcl2mid does not occur at room tempera- ture but is detected when heated above the melting point. In addition, the use of the lithium cation to keep the same ionic strength in a K þ solution favors the formation of the bcl2mid dimer. We also found that the bcl2mid dimer is more stable than the monomer. However, after the bcl2mid monomer is formed in a K þ solution, there is no appreciable structural conversion from the monomer to the dimer detected with addition of Li þ at room temperature. Furthermore, the spectral changes of bcl2mid when transitioning from sodium form to potassium form take place upon K þ titration. The absence of the dimer form for bcl2mid after the direct addition of 150 mM [K þ ] at room temperature suggests that the spectral changes are not due to rapid unfolding and refolding. In addition, this work reveals the conditions that would be useful for NMR studies of G-quadruplexes.