Multiple replacements at position 211 in the alpha subunit of tryptophan synthase as a probe of the folding unit association reaction

Equilibrium and kinetic studies on the folding of a series of amino acid replacements at position 211 in the alpha subunit of tryptophan synthase from Escherichia coli were performed in order to determine the role of this position in the rate-limiting step in folding. Previous studies [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965-2974] have shown that the rate-limiting step corresponds to the association/dissociation of the amino (residues 1-188) and carboxy (residues 189-268) folding units. In terms of the secondary structure, the amino folding unit consists of the first six strands and five alpha helices of this alpha/beta barrel protein. The carboxy folding unit comprises the remaining two strands and three alpha helices; position 211 is in strand 7. Replacement of the wild-type glycine at position 211 with serine, valine, and tryptophan at most alters the rate of dissociation of the folding units; association is not changed significantly. In contrast, glutamic acid and arginine dramatically decelerate and accelerate, respectively, both association and dissociation. The difference in effects is attributed to long-range electrostatic interactions for these charged side chains; steric effects and/or hydrogen bonding play lesser roles. When considered with previous data on replacements at other positions in the alpha subunit [Hurle, M. R., Tweedy, N. B., & Matthews, C. R. (1986) Biochemistry 25, 6356-6360], it is clear that beta strands 6 (in the amino folding unit) and 7 (in the carboxy folding unit and containing position 211) dock late in the folding process.     

Proline isomerization and the slow folding reactions of the alpha subunit of tryptophan synthase from Escherichia coli

Previous studies on the refolding of the alpha subunit of tryptophan synthase from Escherichia coli assigned two slow refolding phases to rate-limiting isomerizations of two 'essential' proline residues, one in each of the two domains of the protein (Matthews, C.R., Crisanti, M.M., Manz, J.T. and Gepner, G.L. (1983) Biochemistry 22, 1445-1452). The double-jump experiment (Brandts, J.F., Halvorson, H.R. and Brennan, M. (1975) Biochemistry 14, 4953-4963) was used to further investigate this phenomenon. The reaction assigned to the carboxyl domain is consistent with the proline isomerization hypothesis. The amino domain process is more rapid than expected for proline isomerization and may reflect another type of slow folding reaction. The results permit a further refinement of the folding model for the alpha subunit and demonstrate the existence of a third unfolded species whose folding is not limited by either of these two reactions.     

Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli

The effects of four single amino acid replacements on the stability and folding of the alpha subunit of tryptophan synthase from Escherichia coli have been investigated by ultraviolet differences spectroscopy. In previous studies [Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586], it had been shown that the urea-induced unfolding at pH 7.8, 25 degrees C, proceeds by the initial unfolding of the less stable carboxyl domain (residues 189-268) followed by the unfolding of the more stable amino domain (residues 1-188). The effects of the Phe-22----Leu, Glu-49----Met, Gly-234----Asp, and Gly-234----Lys mutants on the equilibrium unfolding process can all be understood in terms of the domain unfolding model. With the exception of the Glu-49----Met replacement, the effects on stability are small. In contrast, the effects of three of the four mutations on the kinetics of interconversion of the native form and one of the stable partially folded intermediates are dramatic. The results for the Phe-22----Leu and Gly-234----Asp mutations indicate that these residues play a key role in the rate-limiting step. The Glu-49----Met mutation increases the stability of the native form with respect to that of the intermediate but does not affect the rate-limiting step. The Gly-234----Lys mutation does not affect either the stability or the kinetics of folding for the transition between native and intermediate forms. The changes in stability calculated from the unfolding and refolding rate constants agree quantitatively with those obtained from the equilibrium data. When considered with the results from a previous study on the Gly-211----Glu replacement [Matthews, C. R., Crisanti, M. M., Manz, J. T., & Gepner G. L. (1983) Biochemistry 22, 1445], it can be concluded that the rate-limiting step in the conversion of the intermediate to the native conformation involves either domain association or some other type of molecule-wide phenomenon.     

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. I. Kinetic properties.

A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF. Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.     

Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [L?bermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.     

Quaternary structure of rice nonsymbiotic hemoglobin

Plant nonsymbiotic hemoglobins are hexacoordinate heme proteins found in all plants. Although expression is linked with hypoxic environmental conditions (Taylor, E. R., Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-862), no discrete physiological function has yet been attributed to this family of proteins. The crystal structure of a nonsymbiotic hemoglobin from rice has recently been determined. The crystalline protein is homodimeric and hexacoordinate with two histidine side chains coordinating the heme iron atom. Despite the fact that the amino acids responsible for the subunit interface are relatively conserved among the nonsymbiotic hemoglobins, previous work suggests that this group of proteins might display variability in quaternary structure (Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high pressure liquid chromatography were used to investigate the quaternary structure of rice nonsymbiotic hemoglobin at various states of ligation and oxidation. Additionally, site-directed mutagenesis was used to test the role of several interface amino acids in dimer formation and ligand binding. Results were analyzed in light of possible physiological functions and indicate that the plant nonsymbiotic hemoglobins are not oxygen transport proteins but more closely resemble known oxygen sensors.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions

Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Kinetics of oxygen binding to hemoglobin A

The two-state model [Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118] postulates a single conformational change which, in the case of hemoglobin, has been related to the structural differences between deoxy and ligated hemoglobins [Perutz, M. F. (1979) Nature (London) 228, 726-739]. In its simplest form, the model does not represent satisfactorily either the equilibrium or the kinetics of the hemoglobin-oxygen reaction. The kinetic difficulty is with the rate of dissociation from the T-state, and may be met by assuming a wide difference in behavior between alpha- and beta-subunits. Experiments with Ni-Fe hybrids, however, show almost identical rates of combination with, and dissociation from, the two types of subunit, both of which develop R-like reactions as the pH is raised, the alpha-Fe-subunits at lower pH than the beta-Fe-subunits [Shibayama, N., Yonetani, T., Regan, R. M., and Gibson, Q. H. (1995) Biochemistry 34, 14658-14667]. The reactions of oxygen with hemoglobin A and the effect of pH upon them may be represented by assuming behavior of its subunits similar to that of the Ni-Fe hybrids. In such a scheme, alpha-alpha and beta-beta interactions become important elements in cooperativity, and more than two allosteric states are required, for reconsideration of the structural basis of cooperativity.     

NMR study of the exchange rates of allosterically responsive labile protons in the heme pockets of hemoglobin A.

1H NMR spectroscopy has been used to measure the proximal histidyl labile ring proton (NH) rates of exchange with bulk solvent in the individual subunits of hemoglobin (Hb) A. These protons displayed a substantial decrease in their exchange rates in comparison with related monomeric proteins and exhibited sensitivity to the quarternary state. With the beta subunit NH, the exchange behaviour was similar to an allosterically responsive subset of protons, which have been identified using 1H-3H methods (Englander, J.J., R. Rogero, and S. W. Englander, 1983, J. Mol. Biol. 169:325-344). Assuming similar exchange mechanisms for the two subunits, the NMR data suggested a more flexible alpha than beta subunit in Hb A.     

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.     

Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann)

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)     

The amino-acid sequence of the 2S sulphur-rich proteins from seeds of Brazil nut (Bertholletia excelsa H.B.K.)

Storage proteins of the albumin solubility fraction from seeds of Bertholletia excelsa H.B.K. were separated by reversed-phase high-performance liquid chromatography and their primary structures were determined by gas-phase sequencing on intact polypeptides and on the overlapping tryptic and thermolysin peptides. The 2S storage proteins consist of two subunits linked by disulphide bridges. The large subunit (8.5 kDa) is expressed in at least six different isoforms while the small subunit (3.6 kDa) consists of only one form. These proteins are extremely rich in glutamine, glutamic acid, arginine and the sulphur-containing amino acids cysteine and methionine. One of the variants even contains a sequence of six methionine residues in a row. Comparison with known sequences of 2S proteins of other dicotyledonous plants shows limited but distinct sequence homology. In particular, the positions of the cysteine residues relative to each other appear to be completely conserved, suggesting that tertiary structure constraints imposed by disulphide bridges dominate sequence conservation. It has been proposed that the two subunits of a related protein (the Brassica napus storage protein) is cleaved from a precursor polypeptide [Crouch, M. L., Tenbarge, K. M., Simon, A. E. & Ferl, R. (1983) J. Mol. Appl. Genet. 2,273-283]. The amino acid sequence homology of the Brazil nut protein with the former suggests that a similar protein processing event could occur.     

Identification of sites phosphorylated in bovine cardiac troponin I and troponin T by protein kinase C and comparative substrate activity of synthetic peptides containing the phosphorylation sites

As an extension of our previous reports that cardiac and skeletal muscle troponin I (Tn-I) and troponin T (Tn-T) are excellent substrates for protein kinase C (PKC) (Katoh, N., Wise, B. C., and Kuo, J. F. (1983) Biochem. J. 209, 189-195; Mazzei, G. J., and Kuo, J. F. (1984) Biochem. J. 218, 361-369), we have now determined that PKC phosphorylated serine 43 (and/or serine 45), serine 78, and threonine 144 in the free Tn-I subunit and threonine 190, threonine 199, and threonine 280 in the free Tn-T subunit of bovine cardiac troponin. PKC appeared to phosphorylate the same sites of the subunits present in the form of the troponin complex, as indicated by the similarity in the two-dimensional phosphopeptide maps. Although some of the phosphorylation sites were shared by other classes of protein kinases, PKC exhibited a distinct substrate specificity. It was also noted that phosphorylated serine and threonine residues in Tn-I and Tn-T had neighboring basic amino acid residues separated by 1 or 2 other residues both at the amino and carboxyl termini, in agreement with the conclusion of House et al. (House, C., Wettenhall, R. E. H., and Kemp, B. E. (1987) J. Biol. Chem. 262, 772-777) based upon their studies on other substrate proteins. Several peptides having sequences around the phosphorylating sites have been synthesized. The phosphorylation experiments indicated that these peptides were substrates for PKC, and their relative substrate activity (determined by the ratios of Vmax/Km) compared with other proteins, in descending order, was Tn-I = Tn-I(134-154) greater than Tn-T much greater than histone H1 greater than Tn-I(33-35) approximately Tn-T(268-284) greater than Tn-T(179-198) approximately Tn-T(191-209). It is suggested that PKC phosphorylation of Tn-I and Tn-T could be biologically significant in terms of possible modifications in interactions among the individual contractile protein components as well as the Ca2+ sensitivity and activity of actomyosin ATPase.     

Characterization of the carboxyl-terminal sequences responsible for protein retention in the endoplasmic reticulum.

The COOH-terminal sequence KDEL has been shown to be essential for the retention of several proteins in the lumen of the endoplasmic reticulum (Munro S., and Pelham, H. R. B. (1987) Cell 48, 899-907; Pelham, H. R. B. (1988) EMBO J. 7, 913-918; Mazzarella; R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990) J. Biol. Chem. 265, 1092-1101). We have previously demonstrated that variants to the KDEL retention signal, particularly at the initial two positions of the tetrapeptide, can be made without affecting its ability to direct intracellular retention when appended to the neuropeptide Y precursor (pro-NPY) (Andres, D. A., Dickerson, I. M., and Dixon, J. E. (1990) J. Biol. Chem. 265, 5952-5955). To further investigate the nature of the KDEL retention signal, oligonucleotide-directed mutagenesis and transfection was used to generate stable mouse anterior pituitary AtT-20 cell lines expressing pro-NPY mutants with variants of the KDEL sequence added to their direct carboxyl terminus. Analyses of dibasic processing and indirect immunofluorescent microscopy of AtT-20 subclones were consistent with the retention of the pro-NPY mutants bearing the COOH-terminal extensions QDEL, KEDL, or KDEI within the endoplasmic reticulum. A change in the final amino acid of the tetrapeptide from Leu to Val abolished retention completely, and the peptide hormone was processed and secreted. These results indicate that only a limited number of conservative changes can be made to the final two positions of the tetrapeptide without abolishing activity and suggest a highly specific interaction of the retention signal and the KDEL receptor.     

Formation of non-beta 6.3-helical gramicidin channels between sequence-substituted gramicidin analogues.

Using the linear gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221-234). Generally, the gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87-89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249-5256).     

Alleviation of a defect in protein folding by increasing the rate of subunit assembly

Understanding the nature of protein grammar is critical because amino acid substitutions in some proteins cause misfolding and aggregation of the mutant protein resulting in a disease state. Amino acid substitutions in phage P22 coat protein, known as tsf (temperature-sensitive folding) mutations, cause folding defects that result in aggregation at high temperatures. We have isolated global su (suppressor) amino acid substitutions that alleviate the tsf phenotype in coat protein (Aramli, L. A., and Teschke, C. M. (1999) J. Biol. Chem. 274, 22217-22224). Unexpectedly, we found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates.     

The folding transition state of protein L is extensive with nonnative interactions (and not small and polarized)

Progress in understanding protein folding relies heavily upon an interplay between experiment and theory. In particular, readily interpretable experimental data that can be meaningfully compared to simulations are required. According to standard mutational ? analysis, the transition state for Protein L contains only a single hairpin. However, we demonstrate here using ψ analysis with engineered metal ion binding sites that the transition state is extensive, containing the entire four-stranded β sheet. Underreporting of the structural content of the transition state by ? analysis also occurs for acyl phosphatase [Pandit, A. D., Jha, A., Freed, K. F. &; Sosnick, T. R., (2006). Small proteins fold through transition states with native-like topologies. J. Mol. Biol. 361, 755–770], ubiquitin [Sosnick, T. R., Dothager, R. S. &; Krantz, B. A., (2004). Differences in the folding transition state of ubiquitin indicated by ? and ψ analyses. Proc. Natl Acad. Sci. USA 101, 17377–17382] and BdpA [Baxa, M., Freed, K. F. &; Sosnick, T. R., (2008). Quantifying the structural requirements of the folding transition state of protein A and other systems. J. Mol. Biol. 381, 1362–1381]. The carboxy-terminal hairpin in the transition state of Protein L is found to be nonnative, a significant result that agrees with our Protein Data Bank-based backbone sampling and all-atom simulations. The nonnative character partially explains the failure of accepted experimental and native-centric computational approaches to adequately describe the transition state. Hence, caution is required even when an apparent agreement exists between experiment and theory, thus highlighting the importance of having alternative methods for characterizing transition states.