The binding of adenosine 3',5'-cyclic monophosphate (cAMP) and its nonfunctional analogue, guanosine 3',5'-cyclic monophosphate (cGMP), to the adenosine 3',5'-cyclic monophosphate receptor protein (CRP) from Escherichia coli was investigated by means of fluorescence and isothermal titration calorimetry (ITC) at pH 7.8 and 25 degrees C. A biphasic fluorescence titration curve was observed, confirming the previous observation reported by this laboratory (Heyduk and Lee (1989) Biochemistry 28, 6914-6924). However, the triphasic titration curve obtained from the ITC study suggests that the cAMP binding to CRP is more complicated than the previous conclusion that CRP binds sequentially two molecules of cAMP with negative cooperativity. The binding data can best be represented by a model for two identical interactive high-affinity sites and one low-affinity binding site. Unlike cAMP, the binding of cGMP to CRP exhibits no cooperativity between the high-affinity sites. The effects of mutations on the bindings of cAMP and cGMP to CRP were also investigated. The eight CRP mutants studied were K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L. These sites are located neither in the ligand binding site nor at the subunit interface. The binding was monitored by fluorescence. Although these mutations are at a variety of locations in CRP, the basic mechanism of CRP binding to cyclic nucleotides has not been affected. Two cyclic nucleotide molecules bind to the high-affinity sites of CRP. The cooperativity of cAMP binding is affected by mutation. It ranges from negative to positive cooperativity. The binding of cGMP shows none. With the exception of the T127L mutant, the free energy change for DNA-CRP complex formation increases linearly with increasing free energy change associated with the cooperativity of cAMP binding. This linear relationship implies that the protein molecule modulates the signal in the binding of cAMP, even though the mutation is not directly involved in cAMP or DNA binding. In addition, the significant differences in the amplitude of fluorescent signal indicate that the mutations also affect the surface characteristics of CRP. These results imply that these mutations are not perturbing specific pathways of signal transmission. Instead, these results are more consistent with the concept that CRP exists as an ensemble of native states, the distribution of which can be altered by these mutations.

We have previously shown that two K+ must bind to cause the conformational change in Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase reported by fluorescein 5'-isothiocyanate modification and that the ratio of the macroscopic K+ dissociation constants is 4 [Smirnova, I. N., & Faller, L. D. (1993) J. Biol. Chem. 268, 16120-16123]. Since 4 is the ratio expected for random binding to two identical and independent sites, in this paper we propose a two-equivalent-site mechanism for Na+ as well as K+ binding to sodium pump and control of the conformational change between E1 and E2. The equivalent site mechanism is tested by fitting algebraic equations for the empirical first-order rate constant and for the magnitude of the fluorescence change observed in stopped-flow experiments to an array of data. The estimated rate constants for the conformational change at 15 degrees C are kf = 150 +/- 19 s-1 and kr = 0.13 +/- 0.03 s-1, and the estimated dissociation constants for K+ and Na+ from the E1 conformation of the enzyme are KK = 4.8 +/- 1.2 mM and KNa = 0.38 +/- 0.12 mM, in good agreement with estimates from published binding measurements. Although the possibility of ordered and anticooperative binding cannot be excluded, the equivalent site interpretation is supported by quasi-independent estimates of the macroscopic Na+ dissociation constants that give the ratio 4 predicted for binding to identical, noninteracting sites and by equivalent site interpretations of published transport experiments. Six alternative mechanisms are tested. Noncompetitive binding and mutually exclusive binding of the transported ions can be ruled out. Comparable fits to the data are obtained by three-equivalent-site mechanisms in which enzyme with either two or three K+ bound can undergo the conformational change. Therefore, this study quantitatively supports the conformational hypothesis by showing that a conformational change in sodium pump has the right properties to explain transport.

Gramicidin A analogs, labeled with 13C in the backbone carbonyl groups and the C-2 indole carbons of the tryptophan-11 and tryptophan-13 residues, were synthesized using t-Boc-protected amino acids. The purified analogs were incorporated into phosphatidylcholine bilayers at a 1:15 molar ratio and macroscopically aligned between glass coverslips. The orientations of the labeled groups within the channel were investigated using solid-state NMR and the effect of a monovalent ion (Na+) on the orientation of these groups determined. The presence of sodium ions did not perturb the 13C spectra of the tryptophan carbonyl groups. These results contrast with earlier results in which the Leu-10, Leu-12, and Leu-14 carbonyl groups were found to be significantly affected by the presence of sodium ions and imply that the tryptophan carbonyl groups are not directly involved in ion binding. The channel form of gramicidin A has been demonstrated to be the right-handed form of the beta 6.3 helix: consequently, the tryptophan carbonyls would be directed away from the entrance to the channel and take part in internal hydrogen bonding, so that the presence of cations in the channel would have less effect than on the outer leucine residues. Sodium ions also had no effect on the C-2 indole resonance of the tryptophan side chains. However, a small change was observed in Trp-11 when the ether lipid, ditetradecylphosphatidylcholine, was substituted for the ester lipid, dimyristoylphosphatidylcholine, indicating some sensitivity of the gramicidin side chains to the surrounding lipid.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actinactivated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 on HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (eADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of eADP from S1 but did not affect the rate of EADP release from acto-Sl complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such iiodulation involves a mechanism other than regulation of ADP release from myosin.Cyclic interactions of myosin heads with actin filaments coupled to ATP hydrolysis are the molecular basis of muscle contraction. It is believed that the chemical energy from ATP hydrolysis is transduced into mechanical force and movement through conformational changes in the myosin head. The actomyosin-ATP hydrolysis cycle can be simplified as described by Scheme 1 (1).

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.