Sensitivity of the folding pathway to the details of amino acid sequence

Experimental data on the structure of transition state demonstrate that the proteins with the same topology as a rule have similar folding nuclei (the structured formed part of the transition state). In this review we discuss the experimental works which show that the position of folding nuclei is different among proteins with the same topology. These facts underline that the folding pathway is sensitive to the details of amino-acid sequence.     

Tracing hybrid incompatibilities to single amino acid substitutions

Deleterious interactions among genes cause reductions in fitness of interpopulation hybrids (hybrid breakdown). Identifying genes involved in hybrid breakdown has proven difficult, and few studies have addressed the molecular basis of this widespread phenomenon. Because proper function of the mitochondrial electron transport system (ETS) requires a coadapted set of nuclear and mitochondrial gene products, ETS genes present an attractive system for studying the evolution of coadapted gene complexes within isolated populations and the loss of fitness in interpopulation hybrids. Here we show the effects of single amino acid substitutions in cytochrome c (CYC) on its functional interaction with another ETS protein, cytochrome c oxidase (COX) in the intertidal copepod Tigriopus californicus. The individual and pairwise consequences of three naturally occurring amino acid substitutions in CYC are examined by site-directed mutagenesis and found to differentially effect the rates of CYC oxidation by COX variants from different source populations. In one case, we show that interpopulation hybrid breakdown in COX activity can be attributed to a single naturally occurring amino acid substitution in CYC.     

The amino acid sequence of a Bacillus subtilis phosphoprotein that matches an orfY-tsr coding sequence

Bacillus subtilis contains a 30 kDa protein which was phosphorylated during late vegetative growth and sporulation. The sequence for the N-terminal 16 amino acids was found to be identical to the predicted sequence for the N-terminus of a small open reading frame, orfY, but diverged from the predicted sequence thereafter. The orfY region was resequenced and contained one less adenine residue than previously reported, resulting in an open reading frame from within orfY through the entire coding region for tsr which follows orfY. The predicted orfY-tsr amino acid sequence showed 24% identity to Escherichia coli fructose-1,6-bisphosphate aldolase. Two mutants in the tsr region had 2-5% of wild-type aldolase and the nucleotide sequences showed missense mutations. These results indicate that orfY-tsr encodes aldolase and should be renamed fba1.     

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.     

The amino acid sequence of a hypothalamic peptide, neurotensin.

The amino acid sequence of neurotensin, a hypotensive peptide isolated from acid-acetone extracts of bovine hypothalami, has been established as less than Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Oh. (The nomenclature and symbols follow the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1972) J. Biol. Chem. 247, 977). This was accomplished by sequence analyses performed on the intact peptide as well as its isolated tryptic, chymotryptic, and papain-generated fragments. The results of enzymic hydrolyses were consistent with the specificities of the enzymes used and indicated that all of the amino acids are unsubstituted and in the L configuration. The absence of non-amino acid constituents was further supported by analyses of electrophoretic mobility-molecular weight relationships of neurotensin and its fragments.     

Reversing the amino acid sequence of a dipeptide changes its partition in an aqueous two-phase system

Summary Reversing the amino acid sequence of a dipeptide produces distinct differences in how they partition between a polyethylene glycol/potassium phosphate aqueous two-phase system. The partition coefficients for twenty-three dipeptides in this biphasic system are recorded.     

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

Rhodopseudomonas palustris strain RCB100 degrades 3-chlorobenzoate (3-CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3-CBA and determined its N-terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate-CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3-CBA as a sole carbon source. The aliA gene from the 3-CBA degrading strain differed by a single nucleotide from the aliA gene from strain CGA009, causing the substitution of a serine for a threonine at position 208. Both AliA enzymes, purified as His-tagged fusion proteins, had comparable activities with cyclohexanecarboxylate. However, AliA from the 3-CBA degrading strain was 10-fold more active with 3-CBA (kcat/Km of 4.3 x 10(4) M(-1) s(-1)) than the enzyme from the sequenced strain (kcat/Km 0.32 x 10(4) M(-1) s(-1)). The CGA009 enzyme was not sufficiently active with 3-CBA to complement an RCB100 aliA mutant for growth on this compound. Here, whole genome sequence information enabled us to identify a single nucleotide among 5.4 million nucleotides that contributes to the substrate preference of a coenzyme A ligase.     

Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.     

Prediction of protein folding from amino acid sequence over discrete conformation spaces

Predicting the three-dimensional structure of a protein given only its amino acid sequence is a long-standing goal in computational chemistry. In the thermodynamic approach, one needs a potential function of conformation that resembles the free energy of the real protein to the extent that the global minimum of the potential is attained by the native conformation and no other. In practice, this has never been achieved with certainty because even with greatly simplified representations of the polypeptide chain, there are an astronomical number of local minima to examine. If one chooses instead a protein representation with only a large but manageable number of discrete conformations, then the global preference of the potential for the native can be directly verified. Representing a protein as a walk on a two-dimensional square lattice makes it easy to see that simple functions of the interresidue contacts are sufficient to globally favor a given "native" conformation, as long as it is a compact, globular structure. Explicit representation of the solvent is not required. Another more realistic way to confine the conformational search to a finite set is to draw alternative conformations from fragments of larger proteins having known crystal structure. Then it is possible to construct a simple function of interresidue contacts in three dimensions such that only 8 proteins are required to determine the adjustable parameters, and the native conformations of 37 other proteins are correctly preferred over all alternative conformations. The deduced function favors short-range backbone-backbone contacts regardless of residue type and long-range hydrophobic associations. Interactions over long distances, such as electrostatics, are not required.     

Ribulosebisphosphate carboxylase: amino acid sequence of a peptide bearing the activator carbon dioxide

Ribulosebisphosphate carboxylase is activated by reaction of an activator CO2 to form a carbamate on the epsilon-amino group of a lysyl residue on the large catalytic subunit. This carbamate has been converted to the methoxycarbonyl derivative by treatment of the enzyme with diazomethane as previously reported [Lorimer, G. H., & Miziorko, H. H. (1980) Biochemistry 19, 5321]. Digestion of the methylated enzyme--14CO2 complex with trypsin yielded several radioactive peptides which were purified by using standard chromatographic procedures. Sequence analyses revealed that these peptides had the same sequence: -Gly-Gly-Leu-Asp-Phe5-Thr-Lys-Asp-Asp-Glu10-Asn-Val-Asn-Ser-Gln15-Pro-Phe. Residue 7 was 14C labeled and emerged from the sequencer as the phenylthiohydantoin derivative of N epsilon-(methoxycarbonyl)lysine. The acidic nature of the residues close to the lysine bearing the activator CO2 provides a molecular explanation for the pH and divalent metal ion dependency of the activation reaction. An entirely homologous sequence has been found in the large subunit of the enzyme from Zea mais [McIntosh, L., Poulsen, C., & Bogorad, L. (1980) Nature (London) 288, 556]. The lysyl residue bearing the activator CO2 is 26 residues removed from one of the lysyl residues identified by use of the affinity label N-bromoacetyl-ethanolamine phosphate as being within the active-site domain.     

N-Terminal amino acid sequence analysis indicates that isolated atrial amyloid is derived from atrial natriuretic peptide

Isolated atrial amyloid, the most frequent senile cardiac amyloid type, was chemically analysed. Amyloid fibrils obtained from a patient (NIP) were extracted and the predominant lowmolecular-weight polypeptide (approximately 3.5 kDa, designated ASc₂ NIP) was isolated by size exclusion high performance liquid chromatography in 60% formic acid. N-Terminal amino acid sequence analysis of this polypeptide was identical to that of the atrial natriuretic peptide α-hANP for the first 12 residues determined.     

N-terminal amino acid sequence analysis indicates that isolated atrial amyloid is derived from atrial natriuretic peptide

Isolated atrial amyloid, the most frequent senile cardiac amyloid type, was chemically analysed. Amyloid fibrils obtained from a patient (NIP) were extracted and the predominant low-molecular-weight polypeptide (approximately 3.5 kDa, designated ASc2 NIP) was isolated by size exclusion high performance liquid chromatography in 60% formic acid. N-Terminal amino acid sequence analysis of this polypeptide was identical to that of the atrial natriuretic peptide alpha-hANP for the first 12 residues determined.     

Effect of a single amino acid substitution on the folding of the alpha subunit of tryptophan synthase

The urea-induced unfolding of a missense mutant of the alpha subunit of tryptophan synthase from Escherichia coli involving the replacement of Gly by Glu at position 211 has been monitored by absorbance changes at 286 nm. Like the wild-type protein, the equilibrium unfolding curve demonstrates the presence of one or more stable intermediates. Comparison of these results with those from the wild-type alpha subunit [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] shows that the transition from the native conformation to the stable intermediates is displaced to higher urea concentration in the mutant alpha subunit; however, the transition from the intermediates to the unfolded form is unaffected. Kinetic studies show that the amino acid replacement slows the rate of unfolding by an order of magnitude. The effect on refolding rates is complex. One phase, previously assigned to proline isomerization [Crisanti, M. M., & Matthews, C. R. (1981) Biochemistry 20, 2700], is unaffected by the substitution. The rate of the second phase, which is urea dependent down to about 1 M urea, is slower than the corresponding phase in the wild-type protein by approximately a factor of 2. Below about 1 M urea, the rate of this phase becomes urea independent and identical with that of the wild-type alpha subunit. This change in urea dependence has been ascribed to a change in the nature of the rate-limiting step for this process from one involving folding to one involving proline isomerization. The results support the folding model for the alpha subunit proposed previously [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] and clarify the role of proline isomerization in limiting the rate of folding.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.     

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.     

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation.     

The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C

The pediocin-like antimicrobial peptide leucocin C produced by a strain of Leuconostoc mesenteroides has been purified using a recently developed rapid two-step procedure. The complete and corrected amino acid sequence of the peptide has been determined by Edman degradation of the intact peptide and a C-terminal fragment generated by cleavage with Asp-N endoprotease. Leucocin C contained 43 residues with the following sequence: KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTGGNAGWHN. The molecular weight of leucocin C as determined by mass spectrometry was 4595, which is consistent with the theoretical molecular weight of 4596 calculated from the sequence. Moreover, the molecular weights of the two fragments generated by cleavage with Asp-N were also consistent with the determined sequence.