Sedimentation equilibrium studies.

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.

Structure of mammalian AMPK and its regulation by ADP.

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 processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. 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.

Mechanism of interaction between single-stranded DNA binding protein and DNA.

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 approximately 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 x 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 SSB(2) x dT(70) to be 1.1 microM 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.

Three-dimensional molecular mapping in a microfluidic mixing device using fluorescence lifetime imaging.

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.

Structural analysis reveals an amyloid form of the human papillomavirus type 16 E1--E4 protein and provides a molecular basis for its accumulation.

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.

Hydrodynamic characterization of the SufBC and SufCD complexes and their interaction with fluorescent adenosine nucleotides.

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.

Rapid kinetic techniques.

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.

Structural basis for AMP binding to mammalian AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the gamma domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the gamma domain bind either AMP or Mg.ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg.ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the alpha and/or beta subunits binds to the gamma subunit in the presence of AMP but not when ATP is bound.

Structural studies on Plasmodium vivax merozoite surface protein-1.

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-1(42), is cleaved during red blood cell invasion, causing the majority of the complex to be shed and leaving only a small 15kDa sub-unit, MSP-1(19), on the merozite surface. MSP-1(19) is considered a strong vaccine candidate. We have determined the solution structure of MSP-1(19) 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-1(19) is part of the larger MSP-1(42) 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-1(42) exists as a dimer in solution. MSP-1(19) 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-1(42) exists as a dimer in vivo, secondary processing would cause the dissociation of two tightly linked MSP-1(19) proteins on the merozoite surface just prior to invasion.

Cooperative hydrogen bond interactions in the streptavidin-biotin system.

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 approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) 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.

The kinetic mechanism of the SufC ATPase: the cleavage step is accelerated by SufB.

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 Pi. 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.

Sedimentation equilibrium studies.

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.

The mechanism of Ras GTPase activation by neurofibromin.

Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly.

The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct.

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.

H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein.

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.

Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.

Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine (IATR). The doubly labeled peptides have low fluorescence because of ground-state, noncovalent dimerization of the rhodamines. Cleavage of either peptide by recombinant PfSUB-1 results in dissociation of the rhodamine dimers, which abolishes the self-quenching and consequently leads to an approximately 30-fold increase in the fluorescence. This spectroscopic signal provides a continuous assay of proteolysis, enabling quantitative kinetic measurements to be made, and has also enabled the development of a fluorescence-based assay suitable for use in high-throughput screens for inhibitors of PfSUB-1. The structure of the rhodamine dimer in the 6-IATR-labeled peptide was shown by NMR to be a face-to-face stacking of the xanthene rings. Time-resolved fluorescence measurements suggest that the doubly labeled peptides exist in an equilibrium consisting of rhodamines involved in dimers (closed forms) and rhodamines not involved in dimers (open forms). These data also indicate that the rhodamine dimers fluoresce and that the associated lifetimes are subnanosecond.

Oligomerization and kinetic mechanism of the dynamin GTPase.

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 GTPgammaS are also presented. The possible mechanism by which assembly of dynamin leads to an increase in its GTPase activity is discussed.

MgF(3)(-) as a transition state analog of phosphoryl transfer.

The formation of complexes between small G proteins and certain of their effectors can be facilitated by aluminum fluorides. Solution studies suggest that magnesium may be able to replace aluminum in such complexes. We have determined the crystal structure of RhoA.GDP bound to RhoGAP in the presence of Mg(2+) and F(-) but without Al(3+). The metallofluoride adopts a trigonal planar arrangement instead of the square planar structure of AlF(4)(-). We have confirmed that these crystals contain magnesium and not aluminum by proton-induced X-ray emission spectroscopy. The structure adopted by GDP.MgF(-) possesses the stereochemistry and approximate charge expected for the transition state. We suggest that MgF3(-) may be the reagent of choice for studying phosphoryl transfer reactions.