O-GalNAc incorporation into a cluster acceptor site of three consecutive threonines. Distinct specificity of GalNAc-transferase isoforms.

O-Glycosylation of three consecutive Thr residues in a fluorescein-conjugated peptide PTTTPLK - which mimics a portion of mucin 2 - by four isozymes of UDP-N-acetylgalactosaminyltransferases (pp-GalNAc-T1, T2, T3, or T4) was investigated. Partially glycosylated versions of this peptide, PT*TTPLK, PTTT*PLK, PT*TT*PLK, PTT*T*PLK, PT* degrees TTPLK, and PTTT* degrees PLK (*, N-acetylgalactosamine; degrees, galactose), were also tested. The products were separated by RP-HPLC and characterized by MALDI-TOF MS and peptide sequencing. The first and the third Thr residues act as the peptide's initial glycosylation sites for pp-GalNAc-T4, which were different from the sites for pp-GalNAc-T1 and T2 (the first Thr residue) or T3 (the third Thr residue) shown in our previous report. All pp-GalNAc-T isozymes tested exhibited distinct specificities toward glycopeptides. The most notable findings were: (a) prior incorporation of an N-acetylgalactosamine residue at the third Thr greatly enhanced N-acetylgalactosamine incorporation into the other Thr residues when pp-GalNAc-T2, T3, or T4 were used; (b) the enhancing effect of the N-acetylgalactosamine residue on the third Thr was completely abrogated by galactosylation of this N-acetylgalactosamine; (c) prior incorporation of an N-acetylgalactosamine at the first Thr did not have any enhancing effect; (d) pp-GalNAc-T2 was unique as it transferred N-acetylgalactosamine into the second Thr residue only when N-acetylgalactosamine was attached to the third one.     

Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.     

Regulation of mammalian protein O-mannosylation: preferential amino acid sequence for O-mannose modification

O-mannosyl glycans are important in muscle and brain development. Protein O-mannosyltransferase (POMT) catalyzes the initial step of O-mannosyl glycan biosynthesis. To understand which serine (Ser) and threonine (Thr) residues POMT recognizes for mannosylation, we prepared a series of synthetic peptides based on a mucin-like domain in alpha-dystroglycan (alpha-DG), one of the best known O-mannosylated proteins in mammals. In alpha-DG, the mucin-like domain spans amino acid residues 316 to 489. Two similar peptide sequences, corresponding to residues 401-420 and 336-355, respectively, were strongly mannosylated by POMT, whereas other peptides from alpha-DG and peptides of various mucin tandem repeat regions were poorly mannosylated. Peptides 401-420 and 336-355 contained four and six Ser and Thr residues, respectively. Substitution of Ala residues for the Ser or Thr residues showed that Thr-414 of peptide 401-420 and Thr-351 of peptide 336-355 were prominently modified by O-mannosylation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Edman degradation analysis of the mannosylated peptide 401-420 indicated that Thr-414 was the Thr residue that was most prominently modified by O-mannosylation and that O-mannosylation occurred sequentially rather than at random. Based on these results, we propose a preferred amino acid sequence for mammalian O-mannose modification.     

Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated Ser at P = 0).     

Synthesis, metabolic stability and chemotactic activity of peptide T and its analogues

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD4 present on T4 helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4-8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10(-11)-10(-10) M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4-8)-OH abolished the biological action. In the octapeptide series, whereas the alpha-amino butyric acid (Abu) substitution for Thr4 was well tolerated, the same "slight" structural change at Thr5 or Thr8 was very detrimental. Finally, [D-Asn6]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr4 does not interfere with biological characteristics of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mucin core O-glycosylation is modulated by neighboring residue glycosylation status. Kinetic modeling of the site-specific glycosylation of the apo-porcine submaxillary mucin tandem repeat by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases T1 and T2

The influence of peptide sequence and environment on the initiation and elongation of mucin O-glycosylation is not well understood. The in vivo glycosylation pattern of the porcine submaxillary gland mucin (PSM) tandem repeat containing 31 O-glycosylation sites (Gerken, T. A., Gilmore, M., and Zhang, J. (2002) J. Biol. Chem. 277, 7736-7751) reveals a weak inverse correlation with hydroxyamino acid density (and by inference the density of glycosylation) with the extent of GalNAc glycosylation and core-1 substitution. We now report the time course of the in vitro glycosylation of the apoPSM tandem repeat by recombinant UDP-GalNAc:polypeptide alpha-GalNAc transferases (ppGalNAc transferase) T1 and T2 that confirm these findings. A wide range of glycosylation rates are found, with several residues showing apparent plateaus in glycosylation. An adjustable kinetic model that reduces the first-order rate constants proportional to neighboring glycosylation status, plus or minus three residues of the site of glycosylation, was found to reasonably reproduce the experimental rate data for both transferases, including apparent plateaus in glycosylation. The unique, transferase-specific, positional weighting constants reveal information on the peptide/glycopeptide recognition site for each transferase. Both transferases displayed high sensitivities to neighboring Ser/Thr glycosylation, whereas ppGalNAc T2 displayed additional high sensitivities to the presence of nonglycosylated Ser/Thr residues. This is the first demonstration of the ability to model mucin O-glycosylation kinetics, confirming that under the appropriate conditions neighboring glycosylation status can be a significant factor modulating the first step of mucin O-glycan biosynthesis.     

Distinct orders of GalNAc incorporation into a peptide with consecutive threonines

Mucin O-glycosylation is initiated by a transfer of N-acetyl-d-galactosamine (GalNAc) to Ser and Thr residues in polypeptides with a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). In this paper, four human pp-GalNAc-Ts (pp-GalNAc-T1, T2, T3, and T4) were tested for their preferential orders of GalNAc incorporation into FITC-PTTTPITTTTK, a portion of the tandem repeat of human MUC2. The products were separated by reverse-phase HPLC and characterized by MALDI-TOF MS and peptide sequencing. pp-GalNAc-T1 showed preference for acceptor sites, but the order of the incorporation into these sites seemed to be random. In contrast, the GalNAc incorporation by pp-GalNAc-T2, T3, or T4 was not only site-specific but also according to the specific orders. Furthermore, pp-GalNAc-T2, T3, or T4 had distinct maximum numbers of GalNAc incorporations into this peptide.     

Substrate specificity of a multifunctional calmodulin-dependent protein kinase

The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide analogs. The enzyme phosphorylated a synthetic peptide corresponding to the NH2-terminal 10 residues of glycogen synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylated with a Vmax of 12.5 mumol X min-1 X mg-1 and an apparent Km of 7.5 microM compared to values of 1.2 mumol X min-1 X mg-1 and 3.1 microM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a Km of 4 microM and a Vmax of 5.4 mumol X min-1 X mg-1. The importance of the arginine 3 residues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased Vmax 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substrates and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other "arginine-requiring" protein kinases.     

T178 deletion impairs intermolecular interaction of the peptide Nramp1(164–191)

Natural resistance associated macrophage protein 1 (Nramp1), an integral membrane protein with 12 predicted transmembrane domains (TMs), is a divalent cation transporter associated with infectious and autoimmune diseases. A naturally occurring mutation G169D within TM4 of Nramp1 leads to the loss of function, suggesting potential importance of TM4 for the biological function of the protein. In this study, we determine the three‐dimensional structure and topology of a synthetic peptide, del(T178), corresponding to Nramp1(164‐191) (basically consisting of the putative TM4 of Nramp1) with Thr178 deletion in TFE and SDS micelles using NMR and CD spectroscopic techniques, and compare the results with those of the wildtype peptide. Similarly to the wildtype peptide, the del(T178) peptide still forms an amphiphilic‐like α‐helical structure in both membrane mimics and is embedded in SDS micelles. Differently, whereas the wild‐type peptide forms a helix bundle with the hydrophilic side facing the interior of the bundle, the del(T178) peptide exists as a monomer in the membrane mimics and the hydrophilic side of the helix is located near the interface of SDS micelles. Moreover, a strongly cooperative protonation occurs between intramolecular Asp residues for the del(T178) peptide in SDS micelles, while the cooperative proton binding between intermolecular Asp residues was observed for the wildtype peptide. The difference in the results of the two peptides suggests that the deletion of Thr178 impairs intermolecular interaction of the peptide. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Phosphorylation of the LFA-1 integrin beta2-chain on Thr-758 leads to adhesion, Rac-1/Cdc42 activation, and stimulation of CD69 expression in human T cells

Phosphorylation of the leukocyte function-associated antigen-1 (LFA-1) integrin beta2-chain on Thr-758 occurs after T cell receptor stimulation and leads to 14-3-3 recruitment to the integrin, actin cytoskeleton reorganization, and increased adhesion. Here, we have investigated the signaling effects of beta2 integrin Thr-758 phosphorylation. A penetratin-coupled phospho-Thr-758-beta2 peptide (mimicking the part of the integrin beta-chain surrounding Thr-758) stimulated adhesion of human T cells to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). Additionally, the peptide activated the small GTPases Rac-1 and Cdc42 in T cells. Constitutively active forms of Rac-1 and Cdc42, but not Rho, could compensate for the reduction of cell adhesion to ICAM-1 caused by the T758A mutation in the beta2 integrin. Additionally, the active GTPases salvaged the cell-spreading defect of T758A integrin-transfected cells on coated ICAM-1. A dominant negative form of Cdc42, on the other hand, significantly reduced wild-type beta2 integrin-mediated cell adhesion and spreading. In a T cell stimulation system, the pThr-758 penetratin peptide acted in a similar manner to coated ICAM-1 to increase T cell receptor-induced CD69 expression. These results show that Thr-758-phosphorylated LFA-1 is upstream of Rac-1/Cdc42, cell adhesion, and costimulatory activation of human T cells, thus identifying phosphorylation of Thr-758 in beta2 as a proximal element in LFA-1 signaling.     

Photoaffinity labeling of T4 endonuclease V with a substrate containing a phenyldiazirine derivative.

T4 endonuclease V recognizes thymine photodimers in DNA duplexes and, in a two-step reaction, cleaves the glycosyl linkage of the 5'-side thymidine and the phosphodiester linkage. To determine the amino acid residues responsible for binding thymine photodimers, a photoaffinity reagent, 4-(1-azi-2,2,2-trifluoroethyl)-benzoate, was linked to the aminoalkylphosphonate of a thymine photodimer in a 14-mer duplex. The reactive substrate was treated with the enzyme under UV light (365 nm). The nascent enzyme and the modified enzyme were treated with lysyl endopeptidase, and the peptide maps were compared. Three peptides from the C terminus were found to interact with the reactive oligonucleotide to various extents. The three modified peptides were isolated and analyzed by Edman degradation. The amino acid residues Gly-133, Tyr-129, and Thr-89 were partially linked with the reactive substrate and may be involved in the binding of thymine photodimers.     

Peptide T exhibits a well‐defined structure in fluorinated solvent at low temperature

The structure of Peptide T was determined by solution NMR spectroscopy, under strong structure‐inducing conditions: 40% hexafluoro‐2‐propanol aqueous solution at 5 °C. Under these conditions it was possible to detect medium‐range NOEs for the first time for this peptide. This allowed a much better‐defined structure to be determined for Peptide T in comparison with earlier NMR and computational studies. Peptide structures consistent with the experimental restraints were generated using a restrained MD simulation with a full empirical force field. Residues 4–8 of Peptide T take on a well‐defined structure with a heavy atom RMSD of 0.78 Å. The structure is stabilized by hydrogen bonding to side‐chain oxygen atoms of Thr 4 and Thr 8, as well as backbone hydrogen bonding between residues 5 and 7 that forms this region into a classic γ‐turn. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.

We have identified the phosphorylation sites in monkey p53 as well as specific changes in the phosphorylation state of free and complexed forms of simian virus 40 (SV40) large T antigen (T) and monkey p53 isolate from SV40 lytically infected CV1 cells. Phosphopeptide analyses of free T and p53 (To and p53o) and complexed T and p53 (T+ and p53+) fractions indicated several quantitative increases in the specific phosphorylation of complexed forms of both proteins. The N terminus of monkey p53+ is phosphorylated at Ser-9, Ser-15, Ser-20, either Ser-33 or Ser-37, and at least one of Ser-90 to Ser-99. The C-terminal sites are Ser-315 and Ser-392. On comparing p53+ with p53o, we found that labeling of the two N-terminal phosphotryptic peptides encompassing residues 1 to 20 and 33 to 101 was increased fivefold and that Ser-315 was sevenfold more labeled than was Ser-392. When T+ was compared with To, the N-terminal peptide containing phosphorylation sites Ser-106 through Thr-124 was twofold more labeled, the peptide containing Ser-657 through Ser-679 was sixfold more labeled and contained up to four phosphorylated serine residues, and Ser-639 and Thr-701 appeared unchanged. Overall, T+ molecules appeared to contain 3.5 mol more of labeled phosphate than did To, with the N-terminal peptide appearing fully phosphorylated. The phosphopeptide patterns obtained for lytic T+ and To fractions were nearly identical to those found for wild-type SV40 T (stably complexed with mouse p53) and mutant 5080 T (defective for p53 binding) expressed in transformed C3H10T1/2 cells (L. Tack, C. Cartwright, J. Wright, A. Srinivasan, W. Eckhart, K. Peden, and J. Pipas, J. Virol. 63:3362-3367, 1989). These results indicate that increases in specific phosphorylation sites in both T+ and p53+ correlate with the association of T with p53. The enhanced phosphorylation state may be a consequence of complex formation between T and p53 or reflect an increased affinity of p53 for highly phosphorylated forms of T.     

Distinct residues in the carboxyl tail mediate agonist-induced desensitization and internalization of the human dopamine D1 receptor.

We have shown in a previous study that desensitization and internalization of the human dopamine D(1) receptor following short-term agonist exposure are mediated by temporally and biochemically distinct mechanisms. In the present study, we have used site-directed mutagenesis to remove potential phosphorylation sites in the third intracellular loop and carboxyl tail of the dopamine D(1) receptor to study these processes. Mutant D(1) receptors were stably transfected into Chinese hamster ovary cells, and kinetic parameters were measured. Mutations of Ser/Thr residues to alanine in the carboxyl tail demonstrated that the single substitution of Thr-360 abolished agonist-induced phosphorylation and desensitization of the receptor. Isolated mutation of the adjacent glutamic acid Glu-359 also abolished agonist-induced phosphorylation and desensitization of the receptor. These data suggest that Thr-360 in conjunction with Glu-359 may comprise a motif necessary for GRK2-mediated phosphorylation and desensitization. Agonist-induced internalization was not affected with mutation of either the Thr-360 or the Glu-359 residues. However, receptors with Ser/Thr residues mutated in the distal carboxyl tail (Thr-446, Thr-439, and Ser-431) failed to internalize in response to agonist activation, but were able to desensitize normally. These results indicate that agonist-induced desensitization and internalization are regulated by separate and distinct serine and threonine residues within the carboxyl tail of the human dopamine D(1) receptor.     

Identification of Ser153 in ICL2 of the gonadotropin-releasing hormone (GnRH) receptor as a phosphorylation-independent site for inhibition of Gq coupling

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized alphaT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser153 with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser151, Ser153, and Thr142) or only Ser153 was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser153) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in alphaT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser153 operates through a phosphorylation-independent mechanism.     

Nature of the antigenic determinants of T locus antigens

The nature of the antigenic specificities of several antigens associated with the T/t complex in the mouse were analyzed by means of glycosidase and haptene inhibition studies. Results indicate that on testicular cells sugar residues are involved in at least six different T/t antigenic determinants. The immunodominant sugar appears to be different for each of the specificities. The specificity for the following T/t antigens resides predominantly in the sugars indicated: T:sialic acid; t12:beta-D-galactose; tw32:beta-D-galactose; t0:L-fucose; tw1:N-acetyl-D-galactosamine; tw18:L-fucose. It seems probable that these sugars are found at the terminal reducing ends of the carbohydrate portion of T/t-bearing moleculse. These studies imply that at least some of the genes in the T locus code for glycosyltransferases or regulators of glycosyltransferases which modigy oligosaccharide structures and impart specificity to the T/t antigens by alteration of their terminal sugar residues.     

Complete dissection of the Hb(64-76) determinant using T helper 1, T helper 2 clones, and T cell hybridomas.

We have generated cloned Th1 cells, Th2 cells, and T cell hybridomas specific for the single immunogenic peptide from the beta-chain of murine hemoglobin (Hb(64-76)). The availability of these various types of T cells provided us an unique opportunity to examine and dissect the T cell response to an immunogenic peptide. A panel of altered Hb peptides was made by replacing each amino acid in the Hb peptide (positions 64-76) with a conservative amino acid substitution or an alanine. Although none of the eleven T cell clones and hybridomas tested exhibited the same pattern of reactivity to the substituted Hb peptides, some general features were identified for all T cell responses. The primary T cell contact residue of Hb(64-76) was shown to be asparagine 72. For every Hb(64-76) specific T cell, no activation was observed using a peptide containing the conservative substitution of a glutamine for the asparagine at position 72. The flanking glutamic acid at position 73 was also required for a proliferative response for all of the Th1 and Th2 clones. The Th subtypes were not grossly unique in their responses to the substituted Hb peptides, but exhibited minor differences in fine specificity with the Th1 cells identifying more critical amino acids then did the Th2 cells. For the Th1 cells and also the T cell hybridomas, the phenylalanine at position 71 was critical for a T cell response. Analysis of peptide affinity for IEk molecules indicated that position 71 played a role in peptide binding to MHC. Secondary T cell contact residues, which were important for many but not all of the T cells, were identified at positions 69, 70, and 76. Overall T cell responses were minimally affected by changes in the amino acid residues at positions 64-68, 74, and 75. We have also demonstrated that cloned Th1 cells, Th2 cells and T hybridomas can be generated against the same Hb(64-76) determinant.     

Ice-binding mechanism of winter flounder antifreeze proteins

We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic effect play in ice binding are also highlighted. For the latter it is demonstrated that the surface of ice has a clathratelike structure which favors the partitioning of hydrophobic groups to the surface of ice. It is suggested that mutations that involve the deletion of hydrophobic residues (e.g., the Leu residues) will provide insight into the role the hydrophobic effect plays in partitioning these peptides to the surface of ice.     

Biochemical characterization of the Arabidopsis protein kinase SOS2 that functions in salt tolerance

The Arabidopsis Salt Overly Sensitive 2 (SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway. SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensor Salt Overly Sensitive 3. In this study, we examined some of the biochemical properties of the SOS2 in vitro. To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli. Three constitutively active mutants, SOS2T168D, SOS2T168D Delta F, and SOS2T168D Delta 308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain. These active mutants exhibited a preference for Mn(2+) relative to Mg(2+) and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation. The three enzymes had similar peptide substrate specificity and catalytic efficiency. Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium. The active mutant SOS2T168D Delta 308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation. Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop. Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases.