The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).     

Interactions of human melanocortin 4 receptor with nonpeptide and peptide agonists

Specific interactions of human melanocortin-4 receptor (hMC4R) with its nonpeptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two synthetic, small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane alpha-helices (TM) 2, 3, 6, and 7 (residues Glu(100), Asp(122), Asp(126), Phe(261), His(264), Leu(265), and Leu(288)). In addition, a I129A mutation primarily affected the binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. Three-dimensional models of receptor-ligand complexes with their agonists were generated by distance-geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys(40)-Cys(279), Cys(271)-Cys(277)) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His(6), d-Phe(7), Arg(8), and Trp(9)) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His(6) and Arg(6) of NDP-MSH; their substituted piperidines mimic Trp(9) of the peptide and interact with TM5 and TM6, while the d-Phe aromatic rings of all three agonists contact with Leu(133), Trp(258), and Phe(261) residues.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits

Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme. The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide. Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles. Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions. Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects. alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site. A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

Structural determinant for cold inactivation of rodent L-xylulose reductase

L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.     

Ydj1p二聚体中β14~β15与domain-III分离的拉伸分子动力学模拟研究

分子伴侣Hsp40是一种以二聚体的形式调控非天然多肽折叠的热激蛋白。本文通过拉伸分子动力学研究了酵母Hsp40家族成员Ydj1p二聚体中β14-β15与domain-Ⅲ的分离过程,深入探讨了影响Ydj1p二聚体稳定性的重要残基和相互作用力。研究表明,残基Thr366、Asp368、Cys370、Leu372和Phe375在Ydj1P二聚体的形成过程中发挥着重要的作用。其中,β14-β15中的残基Thr366和Asp368分别通过与domain-Ⅲ内的残基Asp291、Trp292和Trp292、Lys294之间形成的氢键,Asp368通过与domain-Ⅲ内的残基Lys314形成盐桥,Cys370、Leu372和Phe375则是通过与domain-Ⅲ形成疏水作用力来稳定Ydj1p二聚体结构。     

The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases

We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non-crystallographic symmetry averaging. The mol- ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of beta-carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.     

Effects of troponin T mutations in familial hypertrophic cardiomyopathy on regulatory functions of other troponin subunits.

We have previously shown that mutations in troponin T (TnT), which is associated with familial hypertrophic cardiomyopathy (HCM), cause an increase in the Ca(2+) sensitivity and a potentiation of cardiac muscle contraction. To gain further insight into the patho-physiological role of these mutations, four mutations (Arg92Gln, Phe110Ile, Glu244Asp, Arg278Cys) were introduced into recombinant human cardiac TnT, and the mutants were exchanged into isolated porcine cardiac myofibrils. The effects of mutations were tested on maximal ATPase activity, the inhibitory function of troponin I (TnI) in the absence of troponin C (TnC), and the neutralizing function of TnC. Arg92Gln, Phe110Ile, and Glu244Asp markedly impaired the inhibitory function of TnI. Arg278Cys also impaired the inhibitory function of TnI, but the effect was much smaller. Phe110Ile and Glu244Asp markedly enhanced the neutralizing function of TnC and potentiated the maximum ATPase activity. Arg92Gln and Arg278Cys only slightly enhanced the neutralizing function of TnC, and they conferred no potentiation on the maximum ATPase activity. These results indicate that mutations in TnT impair multiple processes of Ca(2+) regulation by troponin, and there are marked differences in the degree of impairment from mutation to mutation.     

Universality and structure of the N-end rule

Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.     

Identification of ligand binding site on RXRγ using molecular docking and dynamics methods

Retinoid X receptors (RXRα, β and γ) are recently known to be cancer chemotherapies targets. The ligand binding domains of RXRs have been crystallized, but the information of RXRγ ligand binding site is not yet available due to the lack of liganded complex. A thorough understanding of the ligand binding sites is essential to study RXRs and may result in cancer therapeutic breakthrough. Thus we aimed to study the RXRγ ligand binding site and find out the differences between the three subtypes. Alignment and molecular simulation were carried out for identifying the RXRγ ligand binding site, characterizing the RXRγ ligand binding mode and comparing the three RXRs. The result has indicated that the RXRγ ligand binding site is defined by helices H5, H10, β-sheet s1 and the end loop. Besides hydrophobic interactions, the ligand 9-cis retinoic acid interacts with RXRγ through a hydrogen bond with Ala106, a salt bridge with Arg95 and the π-π interactions with Phe217 and Phe218. The binding modes exhibit some similarities among RXRs, such as the interactions with Arg95 and Ala106. Nonetheless, owing to the absence of Ile47, Cys48, Ala50, Ala51 and residues 225∼237 in the active site, the binding pocket in RXRγ is two times larger than those of RXRα and RXRβ. Meanwhile, spatial effects of Trp84, Arg95, Ala106, Phe217 and Phe218 help to create a differently shaped binding pocket as compared to those of RXRα and RXRβ. Consequently, the ligand in RXRγ undergoes a “standing” posing which is distinct from the other two RXRs.     

A domain of the manganese-stabilizing protein from Synechococcus elongatus involved in functional binding to photosystem II.

Site-directed mutagenesis was performed to investigate whether the two protease-sensitive sequences Phe(156)-Gly(163) and Arg(184)-Ser(191), of the manganese-stabilizing protein (MSP) from a thermophilic cyanobacterium, Synechococcus elongatus (Motoki, A., Shimazu, T., Hirano, M., and Katoh, S. (1998) Biochim. Biophys. Acta 1365, 492-502), are involved in functional interaction with photosystem II (PSII). The ability of MSP to bind to its functional site on the PSII complex and to reactivate oxygen evolution was dramatically reduced by the substitution of Arg(152), Asp(158), Lys(160), or Arg(162) with uncharged residues, by insertion of a single residue between Phe(156) and Leu(157), or by deletion of Leu(157). Substitution of each of the four charged residues with an identically charged residue showed that the charges at Asp(158), and possibly Lys(160), are important for the electrostatic interaction with PSII. The reactivating ability was also strongly affected by the alteration of Phe(156) to Leu. Replacement of Lys(188), the only strictly conserved charged residue in the Arg(184)-Ser(191) sequence, by Gln had only a marginal effect on the function of MSP. High affinity binding of MSP to PSII was also affected significantly by mutation at Arg(152), which is located in a region (Val(148)-Arg(152)) strictly conserved among the 14 sequences so far reported. These results imply that the Val(148)-Gly(163) sequence, which is well conserved among MSPs from cyanobacteria to higher plants, is a domain of MSP for functional interaction with PSII.     

Investigations on unconventional hydrogen bonds in RNA binding proteins: the role of CH...O=C interactions

We have investigated the roles played by C-H...O=C interactions in RNA binding proteins. There was an average of 78 CH...O=C interactions per protein and also there was an average of one significant CH...O=C interactions for every 6 residues in the 59 RNA binding proteins studied. Main chain-Main chain (MM) CH...O=C interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH...O=C interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH...O=C interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH...O=C interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH...O=C interacting polar amino acid residues were solvent exposed while, majority of the CH...O=C interacting non polar residues were excluded from the solvent. Long and medium-range CH...O=C interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH...O=C interacting residues had a higher conservation score. Significant percentage of CH...O=C interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH...O=C interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Electrostatic interactions in an integral membrane protein

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.     

Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli.

Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.