The reversible formation of protein-protein interactions plays a crucial role in many biological processes. In order to carry out a thorough quantitative characterization of these interactions it is essential to establish the oligomerization state of the individual components first. The sedimentation equilibrium method is ideally suited to perform these studies because it allows a reliable, accurate, and absolute value of the solution molecular weight of a macromolecule to be obtained. This technique is independent of the shape of the macromolecule under investigation and allows the determination of equilibrium constants for a monomer multimer self-associating system.

1. Acid extracts of muscle-fibre preparations from human biopsies incubated with [U-14C]glucose were chromatographically analysed. 2. Radioactivity of fructose was significantly greater in muscle from childhood dystrophy patients and also in female carriers of the disease compared with normal, foetal, neurogenic muscle disease and polymyositis control groups. 3. Measurements of the activity of polyol-NADP oxidoreductase (EC 1.1.1.21) and L-iditol-NAD oxidoreductase (EC 1.1.1.14) in muscle extracts compared with the ability of extracts to dephosphorylate fructose 6-phosphate, indicated that the probable route of fructose formation from glucose is via glucitol. The mean activities of the two specified enzymes in dystrophic muscle showed 13and 3.5-fold increases respectively compared with normal muscle, and smaller increases compared with other controls. 4. Direct comparison of labelling patterns following incubation of muscle-fibre preparations with ~-[U-~~C]glucose, ~-[U-~~C]fructose and ~-[U-'~C]glucitol showed that these three substances are rapidly interconvertible mutual major metabolic products, confirming the glucitol pathway as a major route of fructose formation. 5. [U-14C]fructose is readily metabolized by dystrophic muscle, excluding the possibility of its accumulation being the result of poor utilization. 6 . Inhibition of polyol oxidoreductase in dystrophic muscle preparations by 3,3 I tetramethyleneglutarate drastically reduces the ability of the fibres to utilize [U-14C]glucose, and prevents the formation of [14C]fructose. 7. Some implications of these results are discussed in relation to the pathogenesis of childhood muscular dystrophy. * Present address : Molecular Enzymology Laboratory, Biochemistry Department, Medical School, University of Bristol. Correspondence: Dr D. A. Ellis, Muscular Dystrophy Group of Great Britain Research Laboratory, Department of Pathology, Midland Centre for Neurosurgery and Neurology, Holly Lane, Smethwick, Warley, Worcestershire.

AMP-activated protein kinase (AMPK) has an important role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels, it activates energy-producing pathways and inhibits energy-consuming processes. Here, a role for ADP in regulating AMPK by protecting the enzyme from dephosphorylation is defined, and a crystal structure of the active enzyme containing the kinase domain is presented. A model is proposed for how AMP and ADP regulate AMPK activity. The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes1. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer2,3,4,5,6. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain7: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase8, protects the enzyme against dephosphorylation, as well as causing allosteric activation9. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.

A single-stranded DNA binding protein (SSB), labeled with a fluorophore, interacts with single-stranded DNA (ssDNA), giving a 6-fold increase in fluorescence. The labeled protein is the adduct of the G26C mutant of the homotetrameric SSB from Escherichia coli and a diethylaminocoumarin {N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide}. This adduct can be used to assay production of ssDNA during separation of double-stranded DNA by helicases. To use this probe effectively, as well as to investigate the interaction between ssDNA and SSB, the fluorescent SSB has been used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate. Under conditions where ∼70 base lengths of ssDNA wrap around the tetramer, initial association is relatively simple and rapid, possibly diffusion-controlled. The kinetics are similar for a 70-base length of ssDNA, which binds one tetramer, and poly(dT), which could bind several. Under some conditions (high SSB and/or low ionic strength), a second tetramer binds to each 70-base length, but at a rate 2 orders of magnitude slower than the rate of binding of the first tetramer. Dissociation kinetics are complex and greatly accelerated by the presence of free wild-type SSB. The main route of dissociation of the fluorescent SSB·ssDNA complex is via association first with an additional SSB and then dissociation. Comparison of binding data with different lengths of ssDNA gave no evidence of cooperativity between tetramers. Analytical ultracentrifugation was used to determine the dissociation constant for labeled SSB2·dT70 to be 1.1 μM at a high ionic strength (200 mM NaCl). Shorter lengths of ssDNA were tested for binding: only when the length is reduced to 20 bases is the affinity significantly reduced.

In iron overload conditions, plasma contains non-transferrin bound iron species, collectively referred to as plasma NTBI. These include iron citrate species, some of which are protein bound. Because NTBI is taken into tissues susceptible to iron loading, its removal by chelation is desirable but only partial using standard deferoxamine (DFO) therapy. Speciation plots suggest that, at clinically achievable concentrations, deferiprone (DFP) will shuttle iron onto DFO to form feroxamine (FO), but whether NTBI chelation by DFO is enhanced to therapeutically relevant rates by DFP is unknown. As FO is highly stable, kinetic measurements of FO formation by high-performance liquid chromatography or by stopped-flow spectrometry are achievable. In serum from thalassemia major patients supplemented with 10 microM DFO, FO formation paralleled NTBI removal but never exceeded 50% of potentially available NTBI; approximately one third of NTBI was chelated rapidly but only 15% of the remainder at 20 h. Addition of DFP increased the magnitude of the slower component, with increments in FO formation equivalent to complete NTBI removal by 8 h. This shuttling effect was absent in serum from healthy control subjects, indicating no transferrin iron removal. Studies with iron citrate solutions also showed biphasic chelation by DFO, the slow component being accelerated by the addition of DFP, with optimal enhancement at 30 microM. Physiological concentrations of albumin also enhanced DFO chelation from iron citrate, and the co-addition of DFP further accelerated this effect. We conclude that at clinically relevant concentrations, DFP enhances plasma NTBI chelation with DFO by rapidly accessing and shuttling NTBI fractions that are otherwise only slowly available to DFO.

Fragile-X-related proteins form a family implicated in RNA metabolism. Their sequence is composed of conserved N-terminal and central regions which contain Tudor and KH domains and of a divergent C-terminus with motifs rich in arginine and glycine residues. The most widely studied member of the family is probably FMRP (fragile X mental retardation protein), since absence or mutation of this protein in humans causes fragile X syndrome, the most common cause of inherited mental retardation. Understanding the structural properties of FMRP is essential for correlating it with its functions. The structures of isolated domains of FMRP have been reported, but nothing is yet known with regard to the spatial arrangement of the different modules, partly because of difficulties in producing both the full-length protein and its multidomain fragments in quantities, purities and monodispersity amenable for structural studies. In the present study, we describe how we have produced overlapping recombinant fragments of human FMRP and its paralogues which encompass the evolutionary conserved region. We have studied their behaviour in solution by complementary biochemical and biophysical techniques, identified the regions which promote self-association and determined their overall three-dimensional shape. The present study paves the way to further studies and rationalizes the existing knowledge on the self-association properties of these proteins.

The abundant human papillomavirus (HPV) type 16 E4 protein exists as two distinct structural forms in differentiating epithelial cells. Monomeric full-length 16E1--E4 contains a limited tertiary fold constrained by the N and C termini. N-terminal deletions facilitate the assembly of E1--E4 into amyloid-like fibrils, which bind to thioflavin T. The C-terminal region is highly amyloidogenic, and its deletion abolishes amyloid staining and prevents E1--E4 accumulation. Amyloid-imaging probes can detect 16E1--E4 in biopsy material, as well as 18E1--E4 and 33E1--E4 in monolayer cells, indicating structural conservation. Our results suggest a role for fibril formation in facilitating the accumulation of E1--E4 during HPV infection.

pH-dependent interconversion between ring and chain forms of sultams/sulfonamides derived from conjugates of sulforhodamines with amines, and the associated sulfonamide ionization, have been studied by a combination of equilibrium and kinetic methods. The colorless, ring-closed sultam form is favored at alkaline pH with an apparent pK(a) of 7.37 for the color change of the methylamine conjugate of Sulforhodamine B. The ring-closed form is also favored at very low pH (apparent pK(a) similar to 0.68) by protonation of both diethylamino substituents. The kinetics of interconversion between open and closed forms were measured at 4 degrees C over the pH range 0-13. The observed rate constant ranges over nine orders of magnitude from 4.8 x 10(-4) s(-1) at pH 1 to 2.27 X 10(6) s(-1) at pH >= 12. Above pH 2, the data are accommodated by a mechanism that includes cleavage of the sultam C-N bond in the ring opening step with a sulfonamide anion as the leaving group, and in the reverse reaction, OH--dependent ionization of the sulfonamide at 4.8 (+/- 2.0) x 10(9) M-1 s(-1). Below pH 2, H+-dependent protonation occurs at 1.4 (+/- 0.4) x 10(-3) M-1 s(-1). X-ray crystallography of a related N-methylsultam showed that the labile, endocyclic C-N bond is significantly longer than the exocyclic N-CH3 bond (1.509 angstrom and 1.445 angstrom, respectively). Laser-induced ring opening of the closed form has potential application as an orientation probe of biological macromolecules. (C) Crown Copyright 2008.

The elementary steps in complex biochemical reaction schemes (isomerization, dissociation, and association reactions) ultimately determine how fast any system can react in responding to incoming signals and in adapting to new conditions. Many of these steps have associated rate constants that result in subsecond responses to incoming signals or externally applied changes. This chapter is concerned with the techniques that have been developed to study such rapidly reacting systems in vitro and to determine the values of the rate constants for the individual steps. We focus principally on two classes of techniques: (1) flow techniques, in which two solutions are mixed within a few milliseconds and the ensuing reaction monitored over milliseconds to seconds, and (2) relaxation techniques, in which a small perturbation to an existing equilibrium is applied within a few microseconds and the response of the system is followed over microseconds to hundreds of milliseconds. These reactions are most conveniently monitored by recording the change in some optical signal, such as absorbance or fluorescence. We discuss the instrumentation that is (commercially) available to study fast reactions and describe a number of optical probes (chromophores) that can be used to monitor the changes. We discuss the experimental design appropriate for the different experimental techniques and reaction mechanisms, as well as the fundamental theoretical concepts behind the analysis of the data obtained.

Fluorescence lifetime imaging (FLIM) is used to quantitatively map the concentration of a small molecule in three dimensions in a microfluidic mixing device. The resulting experimental data are compared with computational fluid-dynamics (CFD) simulations. A line-scanning semiconfocal FLIM microscope allows the full mixing profile to be imaged in a single scan with submicrometer resolution over an arbitrary channel length from the point of confluence. Following experimental and CFD optimization, mixing times down to 1.3+/-0.4 ms were achieved with the single-layer microfluidic device.

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.

Plasmodium vivax infection is the second most common cause of malaria throughout the world. Like other Plasmodium species, P. vivax has a large protein complex, MSP-1, located on the merozoite surface. The C-terminal MSP-1 sub-unit, MSP-142, is cleaved during red blood cell invasion, causing the majority of the complex to be shed and leaving only a small 15 kDa sub-unit, MSP-119, on the merozite surface. MSP-119 is considered a strong vaccine candidate. We have determined the solution structure of MSP-119 from P. vivax using nuclear magnetic resonance (NMR) and show that, like in other Plasmodium species, it consists of two EGF-like domains that are oriented head-to-tail. The protein has a flat, disk-like shape with a highly charged surface. When MSP-119 is part of the larger MSP-142 precursor it exists as an independent domain with no stable contacts to the rest of the sub-unit. Gel filtration and analytical ultracentrifugation experiments indicate that P. vivax MSP-142 exists as a dimer in solution. MSP-119 itself is a monomer, however, 35 amino-acids immediately upstream of its N-terminus are sufficient to cause dimerization. Our data suggest that if MSP-142 exists as a dimer in vivo, secondary processing would cause the dissociation of two tightly linked MSP-119 proteins on the merozoite surface just prior to invasion.

Protein products of the suf operon are involved in iron- sulfur metabolism. SufC is an ATPase that can interact with SufB in the absence of nucleotide. We have studied the transient kinetics of the SufC ATPase mechanism using the fluorescent ATP analogue, 2'(3')- O- N- methylanthraniloyl- ATP ( mantATP). mantATP initially binds to SufC weakly. A conformational change of the SufC . mantATP complex then occurs followed by the very slow cleavage of mantATP to mantADP and the rapid release of P-i. In the presence of SufB, the cleavage step is accelerated and the release of mantADP is inhibited. Both of these effects promote the formation of a SufC . mantADP complex. In the absence and presence of SufB, mantADP remains more tightly bound to SufC than mantATP. These studies provide a basis for how the SufB and - C proteins interact in the processes involved in regulating iron- sulfur transfer.

The thermodynamic and structural cooperativity between the Ser45– and D128–biotin hydrogen bonds was measured by calorimetric and X‐ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity ∼2 × 107 times lower than that of wild‐type streptavidin at 25°C. The corresponding reduction in binding free energy (ΔΔG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11‐fold greater than that predicted by a linear combination of the single‐mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128–biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild‐type hydrogen bond between D128–OD and biotin–N2 is thermodynamically stronger than that between S45–OG and biotin–N1.