A propensity scale for type II polyproline helices (PPII): aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline-aromatic interactions

Type II polyproline helices (PPII) are a fundamental secondary structure of proteins, common in globular and nonglobular regions and important in cellular signaling. We developed a propensity scale for PPII using a host-guest system with sequence Ac-GPPXPPGY-NH(2), where X represents any amino acid. We found that proline has the highest PPII propensity, but most other amino acids display significant PPII propensities. The PPII propensity of leucine was the highest of all propensities of non-proline residues. Alanine and residues with linear side chains displayed the next highest PPII propensities. Three classes of residues displayed lower PPII propensities: β-branched amino acids (Thr, Val, and Ile), short amino acids with polar side chains (Asn, protonated Asp, Ser, Thr, and Cys), and aromatic amino acids (Phe, Tyr, and Trp). tert-Leucine particularly disfavored PPII. The basis of the low PPII propensities of aromatic amino acids in this context was significant cis-trans isomerism, with proline-rich peptides containing aromatic residues exhibiting 45-60% cis amide bonds, due to Pro-cis-Pro-aromatic and aromatic-cis-Pro amide bonds.     

13C nuclear magnetic resonance study of the cis-trans isomerism in X-Pro-Pro tripeptides.

13C nuclear magnetic resonance has been used to characterize quantitatively the cis-trans isomerism about both peptide bonds in the tripeptides Ser-Pro-Pro and Arg-Pro-Pro. Detailed pH titration data indicate that the configuration about both peptide bonds is closely linked to titration of the terminal carboxyl group and, to a lesser extent, to titration of the terminal amino group. The Pro2 C-3 resonance has been found particularly useful for interpretation due to its sensitivity to the isomerization about both peptide bonds. Analysis of the probabilities of the trans-trans, cic-cis, cis-trans, and trans-cis isomers in aqueous solution indicates a stability decrease in the order given. Similarities in the isomerization behavior of the two peptides indicate that side chain interactions involving the first residue have very little effect on the observed cis/trans ratios. The sensitivity of the cis/trans ratio to titration of the terminal amino group is most readily explained on the basis of an indirect effect on carbonyl-carbonyl repulsion.     

Contribution of tertiary amides to the conformational stability of collagen triple helices

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides.     

Role of phosphorylation in the conformation of tau peptides implicated in Alzheimer's disease

A series of peptides corresponding to isolated regions of Tau (tau) protein have been synthesized and their conformations determined by (1)H NMR spectroscopy. Immunodominant peptides corresponding to tau(224-240) and a bisphosphorylated derivative in which a single Thr and a single Ser are phosphorylated at positions 231 and 235 respectively, and which are recognized by an Alzheimer's disease-specific monoclonal antibody, were the main focus of the study. The nonphosphorylated peptide adopts essentially a random coil conformation in aqueous solution, but becomes slightly more ordered into beta-type structure as the hydrophobicity of the solvent is increased by adding up to 50% trifluoroethanol (TFE). Similar trends are observed for the bisphosphorylated peptide, with a somewhat stronger tendency to form an extended structure. There is tentative NMR evidence for a small population of species containing a turn at residues 229-231 in the phosphorylated peptide, and this is strongly supported by CD spectroscopy. A proposal that the selection of a bioactive conformation from a disordered solution ensemble may be an important step (in either tubulin binding or in the formation of PHF) is supported by kinetic data on Pro isomerization. A recent study showed that Thr231 phosphorylation affected the rate of prolyl isomerization and abolished tubulin binding. This binding was restored by the action of the prolyl isomerase Pin1. In the current study, we find evidence for the existence of both trans and cis forms of tau peptides in solution but no difference in the equilibrium distribution of cis-trans isomers upon phosphorylation. Increasing hydrophobicity decreases the prevalence of cis forms and increases the major trans conformation of each of the prolines present in these molecules. We also synthesized mutant peptides containing Tyr substitutions preceding the Pro residues and found that phosphorylation of Tyr appears to have an effect on the equilibrium ratio of cis-trans isomerization and decreases the cis content.     

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.     

Conformational preferences and prolyl cis–trans isomerization of phosphorylated Ser/Thr‐Pro motifs

The conformational study on Ac‐pSer‐Pro‐NHMe and Ac‐pThr‐Pro‐NHMe peptides has been carried out using hybrid density functional methods with the implicit solvation reaction field theory at the B3LYP/ 6‐311++G(d,p)//B3LYP/6‐31+G(d) level of theory in the gas phase and in solution (chloroform and water). For both pSer‐Pro and pThr‐Pro peptides in the gas phase and in chloroform, the most preferred conformation has the α‐helical structure for the pSer/pThr residue, the down‐puckered polyproline I structure for the Pro residue, and the cis prolyl peptide bond between the two residues, in which two hydrogen bonds between the phosphate oxygens with the backbone N? H groups seem to play a role. However, the trans conformations that have a single hydrogen bond of the phosphate oxygen with either of two backbone N? H groups become most preferred for both peptides in water. This is because the hydration free energy of the anionic oxygen of the phosphate group is expected to dramatically decrease for the cis conformation upon formation of the hydrogen bond with the backbone N? H groups. These calculated results are consistent with the observations by NMR and IR experiments, suggesting the existence of hydrogen bonds between the charged phosphoryl group and the backbone amide protons in solution. The calculated cis populations of 14.7 and 14.2% and rotational barriers of 19.87 and 20.57 kcal/mol to the cis‐to‐trans isomerization for pSer‐Pro and pThr‐Pro peptides in water, respectively, are consistent with the observed values for pSer‐Pro and pThr‐Pro containing peptides from NMR experiments. However, the hydrogen bond between the prolyl nitrogen and the following amide N? H group, which was suggested to be capable of catalyzing the prolyl isomerization, does not play a role in stabilizing the preferred transition state for the pSer/pThr‐Pro peptides in water. Instead, the amide hydrogen of the NHMe group is involved in a bifurcated hydrogen bond with the anionic oxygen and phosphoester oxygen of the phosphate group. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 330–339, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Local and nonlocal environments around Cis peptides

Although the vast majority of peptide bonds in folded proteins are found in the trans conformation, a small percentage are found in the less energetically favorable cis conformation. Though the mechanism of cis peptide bond formation remains unknown, the role of local aromatics has been emphasized in the literature. This paper presents results from a comprehensive statistical analysis of both the local and nonlocal (i.e., tertiary) environment around cis peptides. In addition to an increased frequency of aromatic residues in the local environment around cis peptides, a number of nonlocal differences in protein secondary and tertiary structure between cis and trans peptides are found: (i) coil regions containing cis peptides are almost twice as long as those without cis peptides and include more Tyr and Pro residues; (ii) cis peptides occur with high frequencies in coil regions near large beta-structures; (iii) there is a nonlocal enrichment of Cys, His, Tyr, and Ser in the tertiary environment surrounding cis peptides when compared to trans peptides; and (iv) on average, cis peptides make fewer medium-range and more long-range contacts than trans peptides do. On the basis of these observations, it is concluded that nonlocal factors play a significant role in cis peptide formation, which has not been fully appreciated previously. An autocatalytic model for cis peptide formation is discussed as are consequences for protein folding.     

Proline isomerization in bovine pancreatic ribonuclease A. 2. Folding conditions

The kinetics of cis-trans isomerization of individual X-Pro peptide groups is used to study the backbone dynamics of bovine pancreatic ribonuclease A (RNase A). We previously developed and validated a fluorescence method for monitoring the cis-trans isomerization of the Tyr92-Pro93 and Asn113-Pro114 peptide groups of RNase A under unfolding conditions [Juminaga, D., Wedemeyer, W. J., and Scheraga, H. A. (1998) Biochemistry 37, 11614-11620]. The essence of this method is to introduce a fluorescent residue (Tyr or Trp) in a position adjacent to the isomerizing proline (if one is not already present) and to eliminate the fluorescence of other such residues adjacent to prolines by mutating them to phenylalanine. Here, we extend this method to observe the cis-trans isomerization of these peptide groups under folding conditions using two site-directed mutants (Y92F and Y115F) of RNase A. Both isomerizations decelerate with increasing concentrations of GdnHCl, with nearly identical m values (1.11 and 1.19 M(-1), respectively) and extrapolated zero-GdnHCl time constants (42 and 32 s, respectively); by contrast, under unfolding conditions, the cis-trans isomerizations of both Pro93 and Pro114 are independent of GdnHCl concentration. Remarkably, the isomerization rates under folding conditions at GdnHCl concentrations above 1 M are significantly slower than those measured under unfolding conditions. The temperature dependence of the Pro114 isomerization under folding conditions is also unusual; whereas Pro93 exhibits an activation energy typical of proline isomerization (19.4 kcal/mol), Pro114 exhibits a sharply reduced activation energy of 5.7 kcal/mol. A structurally plausible model accounts for these results and, in particular, shows that folding conditions strongly accelerate the cis-trans isomerization of both peptide groups to their native cis conformation, suggesting the presence of flickering local structure in their beta-hairpins.     

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.     

Ammonia cleaves polypeptides at asparagine proline bonds.

Polypeptides that contain the sequence Asn-Pro undergo complete cleavage at this amide bond with ammonia. One cleavage product possesses Pro as the new amino terminus and the other Asn or isoAsn as the new C-terminus, the formation of the latter probably arising by way of a cyclic succinimide intermediate. Other Asn-X bonds where X = Tyr, Gln, Ile, Glu, Ala, Gly, Asn or Phe did not exhibit any peptide bond cleavage, whereas when X = Leu, Thr and Ser partial cleavage was observed. Asn residues not involved in chain-cleavage underwent deamidation to Asp as shown by MALDI-ToF mass spectrometry (MS) analysis. The partial conversion of in-chain Asp residues to isoAsp under the reaction conditions was inferred from RP-HPLC and MS analysis of reaction mixtures.     

Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A

Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A.     

Local structure in a tryptic fragment of performic acid oxidized ribonuclease A corresponding to a proposed polypeptide chain-folding initiation site detected by tyrosine fluorescence lifetime and proton magnetic resonance measurements

The effects of proline and X-Pro peptide bond conformations on the fluorescence properties of tyrosine in peptides corresponding to parts of a proposed chain-folding initiation site in bovine pancreatic ribonuclease A are examined by time-resolved and steady-state fluorescence spectroscopy. In peptides with Tyr-Pro sequences, the conformational constraints of proline on a preceding residue result in significant fluorescence quenching for both trans and cis peptide bond conformations. Small peptides containing Pro-Tyr sequences, on the other hand, do not exhibit fluorescence quenching compared to Ac-Tyr-NHMe. Studies of fluorescence decay in the tryptic fragment of performic acid oxidized ribonuclease corresponding to residues 105-124 (i.e., O-T-16) demonstrate the presence of at least two environments of the single tyrosine chromophore (in the sequence Asn113-Pro114-Tyr115). In these two (ensemble-averaged) environments, tyrosine has shorter and longer lifetimes, respectively, than in Ac-Tyr-NHMe. The fluorescence heterogeneity in O-T-16 does not correlate with X-Pro cis/trans conformational heterogeneity that can be detected by nuclear magnetic resonance (NMR) spectroscopy. Instead, the fluorescence heterogeneity in O-T-16 arises from the presence of multiple conformations with the same X-Pro peptide bond conformations which interconvert rapidly on the 1H NMR time scale (tau much less than 1 ms) but are distinguishable on the fluorescence lifetime time scale (tau greater than or equal to 1 ns). From comparisons with the tyrosine fluorescence decay of smaller synthetic peptides, it is concluded that the long-lifetime tyrosine fluorescence component of O-T-16 arises from interactions involving residues outside the Asn113-Pro114-Tyr115-Val116-Pro117 sequence, which either stabilize particular local conformations in the vicinity of Tyr115 or act directly to protect Tyr115 from efficient fluorescence quenching. The short-lifetime component of O-T-16 is also observed for the pentapeptide Ac-Asn-Pro-Tyr-Val-Pro-NHMe. The data provide evidence for a nonrandom polypeptide conformation of O-T-16 under conditions of solvent pH and temperature at which the complete disulfide-intact ribonuclease molecule is fully folded. Implications of this work for the interpretation of fluorescence-detected unfolding experiments are discussed.     

Local control of peptide conformation: Stabilization of cis proline peptide bonds by aromatic proline interactions

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constants, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database. © 1998 John Wiley & Sons, Inc. Biopoly 45: 381–394, 1998     

Impact of cis-proline analogs on peptide conformation

The beta-turn is a common motif in both proteins and peptides and often a recognition site in protein interactions. A beta-turn of four sequential residues reverses the direction of the peptide chain and is classified by the phi and psi backbone torsional angles of residues i + 1 and i + 2. The type VI turn usually contains a proline with a cis-amide bond at residue i + 2. Cis-proline analogs that constrain the peptide to adopt a type VI turn led to peptidomimetics with enhanced activity or metabolic stability. To compare the impact of different analogs on amide cis-trans isomerism and peptide conformation, the conformational preference for the cis-amide bond and the type VI turn was investigated at the MP2/6-31+G** level of theory in water (polarizable continuum water model). Analogs stabilize the cis-amide conformations through different mechanisms: (1) 5-alkylproline, with bulky hydrocarbon substituent on the C(delta) of proline, increases the cis-amide population through steric hindrance between the alkyl substituent and the N-terminal residues; (2) oxaproline or thioproline, the oxazolidine- or thiazolidine-derived proline analog, favors interactions between the dipole of the heterocyclic ring and the preceding carbonyl oxygen; and (3) azaproline, containing a nitrogen atom in place of the C(alpha) of proline, prefers the cis-amide bond by lone-pair repulsion between the alpha-nitrogen and the preceding carbonyl oxygen. Preference for the cis conformation was augmented by combining different modifications within a single proline. Azaproline and its derivatives are most effective in stabilizing cis-amide bonds without introducing additional steric bulk to compromise receptor interactions.     

Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

NMR and CD spectroscopy show that imino acid restriction of the unfolded state leads to efficient folding

Protein folding is determined by molecular features in the unfolded state, as well as the native folded structure. In the unfolded state, imino acids both restrict conformational space and present cis-trans isomerization barriers to folding. Because of its high proline and hydroxyproline content, the collagen triple-helix offers an opportunity to characterize the impact of imino acids on the unfolded state and folding kinetics. Here, NMR and CD spectroscopy are used to characterize the role of imino acids in a triple-helical peptide, T1-892, which contains an 18-residue sequence from type I collagen and a C-terminal (Gly-Pro-Hyp)(4) domain. The replacement of Pro or Hyp by an Ala in the (Gly-Pro-Hyp)(4) region significantly decreases the folding rate at low but not high concentrations, consistent with less efficient nucleation. To understand the molecular basis of the decreased folding rate, changes in the unfolded as well as the folded states of the peptides were characterized. While the trimer states of the peptides are all similar, NMR dynamics studies show monomers with all trans (Gly-Pro-Hyp)(4) are less flexible than monomers containing Pro --> Ala or Hyp --> Ala substitutions. Nucleation requires all trans bonds in the (Gly-Pro-Hyp)(4) domain and the constrained monomer state of the all trans nucleation domain in T1-892 increases its competency to initiate triple-helix formation and illustrates the impact of the unfolded state on folding kinetics.     

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.     

Cis peptide bonds in proteins: residues involved, their conformations, interactions and locations.

An analysis of a non-redundant set of protein structures from the Brookhaven Protein Data Bank has been carried out to find out the residue preference, local conformation, hydrogen bonding and other stabilizing interactions involving cis peptide bonds. This has led to a reclassification of turns mediated by cis peptides, and their average geometrical parameters have been evaluated. The interdependence of the side and main-chain torsion angles of proline rings provided an explanation why such rings in cis peptides are found to have the DOWN puckering. A comparison of cis peptides containing proline and non-proline residues show differences in conformation, location in the secondary structure and in relation to the centre of the molecule, and relative accessibilities of residues. Relevance of the results in mutation studies and the cis-trans isomerization during protein folding is discussed.     

Solution structure of BmKalphaIT01, an alpha-insect toxin from the venom of the Chinese scorpion Buthus martensii Karsch

The solution structure of an alpha-insect toxin from Buthus martensii Karsch, BmKalphaIT01, has been determined by two-dimensional NMR spectroscopy and molecular modeling techniques. Combining the sequence homology comparison and toxicity bioassays, BmKalphaIT01 has been suggested to be a natural mutant of alpha-insect toxins and so can serve as a tool to study the relationship of structure-function among this group of toxins. The overall structure of BmKalphaIT01 shares a common core structure consisting of an alpha-helix packed against a three-stranded antiparallel beta-sheet, which exhibits distinctive local conformations within the loops connecting these secondary structure elements. The solution structure of BmKalphaIT01 features a non-proline cis peptide bond between Asn9 and Tyr10, which is proposed to mediate the spatial closing of the five-residue turn (Gln8-Cys12) and the C-terminal segment (Arg58-His64) to form the NC domain and confer the toxin insect-specific bioactivity. Conformational heterogeneity is observed in the solution of BmKalphaIT01 and could be attributed to the cis-trans isomerization of the peptide bond between residues 9 and 10. The minor conformation of BmKalphaIT01 with a trans peptide bond between Asn9 and Tyr10 may be responsible for its moderate bioactivity against mammals. The cis-trans isomerization of the peptide bond between residues 9 and 10 may be the structural basis of dual pharmacological activities of alpha-insect and alpha-like scorpion toxins, which is supported by the fact that conformational heterogeneity occurs in the solution structures of LqhalphaIT, LqqIII, and LqhIII and by comparison of the solution structure of BmKalphaIT01 with those of some relevant alpha-type toxins.