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... [.sup.31]P data GTP samples in varied salt concentrations or pHs. From chemical shifts and [T.sub.1], [T.sub.2] relaxations, it is concluded that the interaction strengths of cations with GTP is in the order of C[a.sup.2+] > M[g.sup.2+] > L[i.sup.+] > N[a.sup.+] for the cations, and in the order of [beta] > [gamma] > [alpha] for the phosphate groups; An increased sample pH causes deprotonation of the [beta] and [gamma] phosphates with enhanced cations' affinities, as indicated by significant chemical shift changes, while the chemical shift of [alpha] phosphate is essentially unaffected by varying pH. These results will aid in exploring the mechanism of GTP-cation interactions, and fully understanding the effects of cations on GTP's stabilization and its biological activities. Key Words: [.sup.31]P NMR; chemical shifts; relaxations; GTP-cation interactions

INTRODUCTION

Guanosine 5'-triphosphate (GTP) is involved in many biological activities, such as the citric acid cycle and the activation of guanine nucleotide-binding proteins (G-proteins), which typically switch between active GTP- and inactive GDP- bound states to regulate a variety of cellular processes including cell growth, apoptosis and differentiation, etc. (1-4). Regulation of cations' binding to GTP is essential for the stabilization of GTP and GTP-mediated functions (5-7). For example, N[a.sup.+] and L[i.sup.+] ions have been shown to induce an inhibition of GTP-stimulated protein activities in vitro (8,9). M[g.sup.2+] is believed to play two distinct roles in Ras-GTP system, i.e. as a conformational regulator through its interactions with the substrate and as a key element for the GTP hydrolysis, while other divalent cations such as C[a.sup.2+] may have no such effect (10), (11).

Nuclear magnetic resonance (NMR) has been widely used in the cation-bound GTP or GTP-bound protein systems to obtain various structural and dynamic information, such as the structural changes of proteins induced by bound GTP or GTP analogs, the mechanism of G-protein activation, the effects of cations on GTP's affinity to proteins, the process of GTP hydrolysis to GDP, and the competitive bindings of different cations to the nucleotides (12-21).

In this publication, we present [.sup.31]P NMR spectroscopy of GTP in varied salt concentrations and pHs. The [.sup.31]P chemical shift and [T.sub.1], [T.sub.2] relaxation data are correlated to characterize the interactions between GTP and different cations (L[i.sup.+], N[a.sup.+], M[g.sup.2+], C[a.sup.2+]), and their affinities for the [alpha], [beta], and [gamma] sites of GTP (see Figure 1). Some structural and dynamic features of GTP in relation to cations' bindings are analyzed.

[FIGURE 1 OMITTED]

MATERIALS AND METHODS

GTP and all other chemicals were purchased from Fisher Scientific/Acros Organics (New Jersey, USA), which were of analytical purity and used without further purification. NMR samples were prepared by dissolving GTP in [D.sub.2]O for the purpose of field lock. To study clear effects of cations on GTP, each of the salts LiCl, NaCl, MGC[l.sub.2] or CaC[l.sub.2] was dissolved in 10 mM GTP solutions with varied salt concentrations in the range of 10 - 500 mM. To study clear effects of pH variation on GTP, the sample pHs were adjusted in the range of pH 2.5 - 10.5 using 1 M NaOH and HCl solutions and were monitored by a pH meter (Denver Instrument).

[.sup.31]P NMR experiments were conducted at room temperature, using a Varian mercury-200 spectrometer operating at spectral frequency 81.015 MHz, and a 4-nucleus auto-NMR probe (suited for [.sup.1]H, [.sup.19]F, [.sup.13]C, [.sup.31]P) with 5 mm sample tubes. The 1D spectra were acquired with typically 14.5 [micro]s of [pi]/2 pulse length, 960...

NOTE: All illustrations and photos have been removed from this article.



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