Mechanical force modifies the free-energy surface of chemical reactions, often enabling thermodynamically unfavoured reaction pathways. Most of our molecular understanding of force-induced reactivity is restricted to the irreversible homolytic scission of covalent bonds and ring-opening in polymer mechanophores. Whether mechanical force can by-pass thermodynamically locked reactivity in heterolytic bimolecular reactions and how this impacts the reaction reversibility remains poorly understood. Using single-molecule force-clamp spectroscopy, here we show that mechanical force promotes the thermodynamically disfavored S N 2 cleavage of an individual protein disulfide bond by poor nucleophilic organic thiols. Upon force removal, the transition from the resulting high-energy unstable mixed disulfide product back to the initial, low-energy disulfide bond reactant becomes suddenly spontaneous, rendering the reaction fully reversible. By rationally varying the nucleophilicity of a series of small thiols, we demonstrate how force-regulated chemical kinetics can be finely coupled with thermodynamics to predict and modulate the reversibility of bimolecular mechanochemical reactions.

A fluorescent reagentless biosensor for inorganic phosphate (P i ), based on the E. coli PstS phosphate binding protein, was redesigned to allow measurements of higher P i concentrations and at low, substoichiometric concentrations of biosensor.This was achieved by weakening P i binding of the previous biosensor, and different approaches are described that could enable this change in properties.The readout, providing response to the P i concentration, is delivered by tetramethylrhodamine fluorescence.In addition to two cysteine mutations for rhodamine labeling at positions 17 and 197, the final variant had an I76G mutation in the hinge region between the two lobes that make up the protein.Upon P i binding, the lobes rotate on this hinge and the mutation on the hinge lowers affinity ∼200-fold, with a dissociation constant now in the tens to hundreds micromolar range, depending on solution conditions.The signal change on P i binding was up to 9-fold, depending on pH.The suitability of the biosensor for steady-state ATPase assays was demonstrated with low biosensor usage and its advantage in ability to cope with P i contamination.

Two forms of biosensor for inorganic phosphate (Pi) have been developed, based on the phosphate binding protein of Escherichia coli, and are now widely used for time-resolved measurements of enzymes that produce Pi. One form is an adduct with a single coumarin fluorophore. For the second version, two cysteine mutations were introduced and labeled with a rhodamine. When physically close to each other and appropriately orientated, two rhodamine dyes can interact to form a non-covalent dimer. In this state they have little or no fluorescence, unlike the high fluorescence intensity of monomeric rhodamine. The labeling sites were so placed that the distance and orientation between the rhodamines changes with the conformation change associated with Pi binding. This movement alters the extent of interaction between rhodamines and gives a large fluorescence increase as Pi binds. In both these forms, Pi binds rapidly and tightly, making them good probes for rapid reaction assays, where all Pi can be bound to the biosensor: that is, the probe must be at least stoichiometric with the Pi. The rhodamine version has a large advantage over coumarin in terms of sensitivity and photobleaching. However, to make the probe more useful for different assay formats, such as high-throughput, the biosensor would best be used sub-stoichiometrically. This is more efficient in terms of usage amounts and has the potential of measuring over a wider range of Pi concentrations. To achieve this, several strategies were examined to locate mutations on this two domain protein to weaken the Pi binding while maintaining the fluorescence response.

Bacteria, as well as the plastid organelles of algae and higher plants, utilize proteins of the suf operon. These are involved in Fe‐S cluster assembly, particularly under conditions of iron limitation or oxidative stress. Genetic experiments in some organisms found that the ATPase SufC is essential, though its role in Fe‐S biogenesis remains unclear. To ascertain how interactions with other individual Suf proteins affect the activity of SufC we coexpressed it with either SufB or SufD from Thermotoga maritima and purified the resulting SufBC and SufCD complexes. Analytical ultracentrifuge and multiangle light‐scattering measurements showed that the SufBC complex exists in solution as the tetrameric SufB 2 C 2 species, whereas SufCD exists as an equilibrium mixture of SufCD and SufC 2 D 2 . Transient kinetic studies of the complexes were made using fluorescent 2′(3′)‐ O ‐( N ‐methylanthraniloyl‐(mant) analogues of ATP and ADP. Both SufBC and SufCD bound mantATP and mantADP much more tightly than does SufC alone. Compared to the cleavage step of the mantATPase of SufC alone, that of SufBC was accelerated 180‐fold and that of SufCD only fivefold. Given that SufB and SufD have 20% sequence identity and similar predicted secondary structures, the different hydrodynamic properties and kinetic mechanisms of the two complexes are discussed.

MP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. This crystal structure of the trimeric regulatory fragment of mammalian AMPK reveals the modes of AMP and ATP binding.

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2'(3')-O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2'-O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k(cat) 0.07 s(-1); however, 3'-O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k(cat) was 0.03 s(-1). Cy3-EDA-ADP.S1 complexes with vanadate (V(i)) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3'-O-Cy3-EDA-ADP.S1.V(i) complex fluorescence doubled and anisotropy increased to 0.372; for the 2'-O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2'-O- and 3'-O-Cy3-EDA-ADP.S1.V(i) complexes, 0.9 and 1.85 ns, compare with approximately 0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP.S1.V(i) complexes, but the 2'-O-isomer only has also a 0.2-ns component. Thus, when bound, 3'-O-Cy3-EDA-ADP's fluorescence is twofold brighter with motion more restricted and turnover slower than the 2'-O-isomer; these data are relevant for applications of these analogs in single molecule studies.

Dynamin is a large molecular weight GTPase. Amongst other biological processes, it is involved in clathrin-dependent endocytosis. It can self-assemble or assemble on other macromolecular structures that result in an increase in its GTPase activity. Its role in endocytosis has been variously attributed to being a force-generating enzyme or a signalling protein. Here we review evidence for the oligomeric state of dynamin at high and low ionic strength conditions. We also review work on the elementary processes of the dynamin GTPase at high ionic strength and compare these to the ATPase of the force-generating protein myosin and the GTPase of the signalling protein Ras. New data on the interaction of dynamin with a fluorescent derivative of GTPγS are also presented. The possible mechanism by which assembly of dynamin leads to an increase in its GTPase activity is discussed.

Commercially-available sulforhodamine sulfonyl chlorides contain two isomeric monosulfonyl chlorides. Conjugates of these isomers with amines have different properties because the sulfonamide formed from one isomer can undergo ring-closure to a colorless sultam. This chemistry has been examined for a model conjugate with methylamine and for a bioconjugate with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP. The interaction of each isomer of the latter conjugates with myosin subfragment 1 has been characterized. Significant differences between the two isomers are observed in these interactions.

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

Dynamin II is a 98 kDa protein (870 amino acids) required fur the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required or its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling, This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 mu M. Cleavage of mantdGTP then occurs to mantdGDP and P-i followed by the rapid release of mantdGDP and P-i. No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in Mujumdar et al. (1993, Bioconjug. Chem. 4:105–111)) with 2′(3′)-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2′- and 3′-O-isomers, the interconversion rate of which was 30[OH−] s−1 (0.016h−1 at pH 7.1) at 22°C. Macroscopic studies showed that 2′(3′)-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2′- and 3′-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.

The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. Formation of the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical hydrolysis and by an EF-Tu.GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex. These spectral differences disappeared when excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 A, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)