Multisite Protein Research Articles

Multisite protein modifications and their determination by mass spectrometry

Multisite protein modifications and their determination by mass spectrometry

An integrated strategy for identification and relative quantification of site‐specific protein phosphorylation using liquid chromatography coupled to MS2/MS3

Reversible and differential multisite protein phosphorylation is an important mechanism controlling the activity of cellular proteins. Here we describe a robust and highly selective approach for the identification and relative quantification of site-specific phosphorylation events. This integrated strategy has three major parts: visualisation of phosphorylated proteins using fluorescently stained polyacrylamide gels, determination of the phosphorylation site(s) using automatic MS3 triggered by the loss of phosphoric acid, and relative quantification of phosphorylation by integrating MS2- and MS3-extracted ion traces using a fast-scanning, linear ion trap mass spectrometer. As a test case, recombinant sucrose-phosphate synthase (SPS) from Arabidopsis thaliana (At5g1110) was used for identification and quantification of site-specific phosphorylation. The identified phosphorylation site of the actively expressed protein coincides with the major regulatory in vivo phosphorylation site in spinach SPS. Site-specific differential in vitro phosphorylation of native protein was demonstrated after incubation of the recombinant protein with cold-adapted plant leaf extracts from A. thaliana, suggesting regulatory phosphorylation events of this key enzyme under stress response.

Multisite protein phosphorylation makes a good threshold but can be a poor switch

Phosphorylation and dephosphorylation play a fundamental role in eukaryotic signaling. Some 30% of proteins are phosphorylated at any time, many on multiple sites, raising the question of how the cellular phosphorylation state is regulated. Previous work for one and two phosphorylation sites has revealed mechanisms, such as distributive phosphorylation, for switch-like regulation of maximally phosphorylated phosphoforms. These insights have led to the influential view that more phosphorylation sites leads to steeper switching, as proposed for substrates like cyclin E and the cyclin-dependent kinase inhibitor Sic1. An analytical study of the ordered distributive case reveals a more complex story. Multisite phosphorylation creates an efficient threshold: The proportion of maximally phosphorylated substrate is maintained close to 0 when the ratio of kinase to phosphatase activity lies below a suitable threshold, and this threshold increases with increasing numbers of sites, n. However, above the threshold, the response may not always abruptly switch between 0 and 1, as would be the case for an efficient switch, but may increase in a gradual manner, which becomes more hyperbolic with increasing n. Abrupt switching cannot be attributed merely to n being large. We point out that conventional measures of ultrasensitivity must be modified to discriminate between thresholding and switching; we discuss additional factors that influence switching efficiency and suggest new directions for experimental investigation.

Differential Multisite Phosphorylation of the Trehalose-6-phosphate Synthase Gene Family in Arabidopsis thaliana

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.

Further examination of a “concerted cluster” model of multivalent affinity heterogeneous adsorption of lactate dehydrogenase to Cibacron blue immobilised on cellulose

A multivalent affinity model, termed the “concerted cluster” model, envisages interactions between a multisite protein and discrete and permanent sets of single paired immobilised ligands, requiring a rigid support matrix. Extensive data on the binding of rabbit muscle lactate dehydrogenase to Cibacron Blue immobilised on cellulose, collected at several immobilised-dye concentrations, gave biphasic Scatchard plots, and were interpreted quantitatively in terms of single and paired ligands. The data were fitted by a least-squares method to model equations. The concentrations of single and paired ligands, and the stoichiometric association constant for single ligands, were in agreement with literature values and model predictions. However, the stoichiometric association constant for ligand pairs was considerably smaller than predicted, indicating less cooperativity within a ligand pair than expected. Nevertheless these results support the hypothesis that cellulose (unlike agarose) provides a sufficiently rigid matrix for permanent ligand clusters to exist.

Heterogeneous binding of aldolase to phosphocellulose: Interpretation in terms of a “concerted cluster” model of multivalent affinity

It has been suggested that when multi-site protein molecules bind to immobilised ligands on an inflexible matrix, they should encounter discrete sets of single and clustered ligands, binding to clusters being concerted and very tight [R.J. Yon, J. Chromatogr., 457 (1988) 13–23]. To test this model, the aldolase-phosphocellulose interaction was re-examined at low protein concentrations, with and without the presence of soluble ligands. In all cases the data plotted as non-linear (concave upwards) Scatchard plots, indicating at least two populations of adsorption sites, while soluble ligands produced competitive effects as expected. When fitted to a 2-population Langmuir model based on the concerted concept, the data suggested that (a) a very small proportion (about 0.3%) of the total immobilised phosphate was accessible as matrix ligand; (b) of this, about 8% comprises accessible pairs; (c) the matrix ligand was non-randomly distributed within the actual matrix volume; (d) affinity constans for soluble ligands were close to their published values, and (e) the effective matrix ligand is a biphosphate structure in phosphocellulose. It is suggested that the concerted-cluster model may be valid for an affinity system based on a “hard” matrix such as cellulose.

Co-operative cluster model for multivalent affinity interactions involving rigid matrices

A rapid-equilibrium model of affinity partitioning of multi-site proteins is described, that applies when long-range translatory movement of matrix-ligand groups is prevented by a rigid matrix. Two essential postulates are made: (1) the matrix ligands are distributed singly or in clusters within spherical bounds of the size of the protein molecule, and may be treated as a Poisson distribution; (2) due to the proximity of matrix ligands, binding of a protein molecule to a cluster is highly co-operative. Equations are derived that allow the predicted partitioning and chromatographic behaviour of this model to be examined and its properties described. Published data on the interaction of aldolase and phosphocellulose are re-interpreted in terms of the new theory.

The interpretation of ligand displacement experiments: Calculations for multisite acceptors

A method is described for calculating the degree of competition for binding between two ligands which are bound at any number of site classes on a binding protein from a generalization of the equilibrium competitive binding equations, the protein's binding parameters for each of the ligands, and the total protein and ligand concentrations. Theoretical displacement curves thus obtained for each of the possible competitive binding models with a multisite protein can then be compared with experimentally determined ligand displacements in order to find which model is most realistic or if measured displacements are due rather to negative cooperativity effects. The binding parameters used for the calculations have a statistical error attached to them, since they have been obtained experimentally, so here we also propose a method for calculating the standard deviations of the theoretical displacement curves deriving from these errors. This permits the use of statistical hypothesis testing in the comparison of theoretical and experimental results. An example is shown in which this method permits the verification that two drugs (phenylbutazone and azapropazone) are both bound by the same high- and low-affinity sites of a protein (α-fetoprotein).

Methyl 4- O-(4-α- d-glucopyranosyloxy-4-methoxybutyl)-α- d-glucopyranoside, a modified oligosaccharide for studying the interactions of carbohydrates with multi-site proteins

Methyl 4- O-(4-α- d-glucopyranosyloxy-4-methoxybutyl)-α- d-glucopyranoside ( 9) was synthesised by transacetalation from methyl 2,3,6-tri- O-acetyl-4- O-(4,4-di-methoxybutyl)-α- d-glucopyranoside and trimethylsilyl 2,3,4,6-tetra- O-acetyl-α- d-glucopyranoside followed by removal of the blocking groups. Compound 9, which is methyl α-maltotrioside modified by replacing the middle d-glucosyl residue with an acyclic spacer, competitively inhibits the hydrolysis of p-nitrophenyl α-maltotrioside by porcine alpha-amylase.

A quantitative study of the biospecific desorption of rat liver (M4) lactate dehydrogenase from 10-carboxydecylamino-Sepharose. Determination of the number of ligand-binding sites blocked on adsorption

1. The theory of Nichol, Ogston, Winzor & Sawyer [(1974) Biochem. J. 143, 435-443] for quantitative affinity chromatography, when adapted for use with a non-specific column from which a multi-site protein can be specifically desorbed by its free ligand, permits determination of the concentration of adsorption sites on the column, their adsorptive affinity (as an association constant) and either the intrinsic (site) constant for ligand-binding to the protein or an 'occlusion coefficient' (defined as the number of ligand-binding sites blocked on adsorption), one of which must be known. 2. The theory has been applied to the NADH-specific desorption of rat liver M4 lactate dehydrogenase from 10-carboxydecylamino-Sepharose. It suggests that most of the enzyme molecules are adsorbed with at least two NADH-binding sites blocked, indicating an extensive adsorption interface in relation to the protein surface. Other chromatographic parameters were also determined for the system. 3. Among topics discussed are (a) factors affecting the experimentally determined value for the number of blocked sites, (b) the nature of the adsorption sites on the column and (c) the similarity of the analysis to that for determining Hill coefficients, and other possible applications.

Mixed and uniform co-operativity of ligand binding to multisite proteins: the co-operativity types allowed by the adair equation and conditions for them

It is proved for the first time that the macroscopic co-operativity of binding to a protein with q binding sites may change signs over a single binding curve any number of times from 0 to ( q-2), but no more than ( q-2). n changes of sign of macroscopic co-operativity requires as a necessary condition at least n changes of sign of microscopic co-operativity, but this necessary condition is not a sufficient one. The necessary and sufficient condition that decides whether there are two changes of sign in a four-site protein is obtained. There are no changes when K 1 K 3( K 2- K 4)+ K 1 K 2( K 3- K 2)+ K 2 K 3( K 4- K 3) is positive, and two changes when it is negative, presuming the above mentioned necessary conditions to be satisfied. The K's of this formula are the “intrinsic” per-site Adair constants. As a result, the conditions for all six co-operativity types possible with a four-site protein are now known.

Multisite proteins and randomly coiled polymeric ligands. Chain length dependence of binding constants.

A model mechanism was developed for the binding of a rigid multisite protein with a randomly coiled multivalent ligand. Probabilities of the formation of chain loops between sites located at given distances at the protein were calculated by an extension of the concept of ring closure in coiled chain molecules. Expressions were derived for the dependence of overall equilibrium quantities, such as the binding constant between the protein and the ligand, on intrinsic parameters such as intrinsic binding constants, number of sites at the protein and their distances and on the chain length of the polymeric ligand. A pronounced chain length dependence of the overall binding constant was predicted even at chain lengths much longer than the size of the protein. Such a dependence was previously observed for the enzyme prolyl hydroxylase which acts on polymeric substrates like (ProProGly)n. This so far unexplained feature is quantitatively described by the model mechanism which is believed to be applicable to many other interactions of biological importance.