Logical analysis of the mechanism of protein folding II. The nucleation process

Regions of secondary structure are predicted, without using information about the conformation of the protein itself, and compared with crystallographic assignments for seven proteins of recently published sequence and conformation (Table 1). It is observed in Table 3 that the prediction of helices is good (78.7% for %cor.ass.3), except for proteins having large antiparallel pleated sheets, and the prediction of β-structure is quite good (51.2% for %cor.ass.3) except for helix-rich proteins.The prediction of secondary structure from sequence, and a survey of all protein structures analysed so far by X-ray crystallography, suggest that nuceleation starts in almost all cases from interactions in the medium range between the regions having helical potential (α-candidate) and β-structural potential (β-candidate), which are very close to each other but separated by at least three hydrophilic or neutral residues in four consecutive residues on the polypeptide chain. Predictability of loops or turns is enhanced to 71.3% (%cor.ass.2) from 64.4% by taking into account the contiguous α-β interactions. Such a medium-range interaction is called here a probable nucleus. There are a lot of nuclei in large proteins such as carboxypeptidase Aα, while there exists at least one in small proteins like the trypsin inhibitor, Moreover, such an interaction could be a transitionary state towards a helix-rich protein, and towards a helix-deficient protein having a large antiparallel pleated sheet β-structure as well.The analysis of the relation between probable nuclei with regard to their mutual spatial proximity strongly suggests that the topological pathway of the polypeptide chain in three-dimensional space might be decided by the long-range interactions between an α-candidate and a β-candidate. An empirical rule is observed that almost all parallel pleated sheets are accompanied by helices in their neighbourhood. An accumulation of chemical facts, such as complementation experiments, combinations of disulphide bonds, etc., seems also to be elucidated by the proposed mechanism of protein folding.     

Structural principles of the globular organization of protein chains. A stereochemical theory of globular protein secondary structure

The paper reveals the types of amino acid sequences of polypeptide chain regions of globular protein which form a regular (α or β) or irregular conformation in the native globule. The study was made taking into account general “architectural” principles of packing of polypeptide chains in globular proteins and considering the interactions of proteins with water molecules. An a priori theory is developed which permits the identification, in good agreement with experiment, of α-helical and β-structural regions in globular proteins from their primary structure.     

Conformational prediction and circular dichroism studies on the lac repressor

Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.     

The Val-210-Ile pathogenic Creutzfeldt–Jakob disease mutation increases both the helical and aggregation propensities of a sequence corresponding to helix-3 of PrPC

A peptide corresponding to the third helical region within the PrP^C protein, from residues 198 to 218 (helix-3), was synthesised with and without the familial 210-Val to Ile Creutzfeldt–Jakob disease mutation. The NMR structure of PrP^C predicts no global variation in stability for this mutation, indicating that local sequence rather than global structural factors are involved in the pathological effects of this mutation. ¹H NMR analysis of peptides with and without this mutation indicated that it had no significant effect on local helical structure. Temperature denaturation studies monitored by CD showed that the mutation increased the helical content within this region (helical propensity), but did not stabilise the helix toward denaturation (helical stability). Aggregation data indicated that, in addition to increasing helical propensity, this mutation increased the aggregation propensity of this sequence. CD and NMR data indicate that helical interactions, stabilised by the Val-210-Ile mutation, may precede the formation of β-sheet aggregates in this peptide sequence. Therefore, this pathological mutation probably does not facilitate PrP^C to PrP^Sc conversion by directly destabilising the helical structure of PrP^C, but may preferentially stabilise PrP^Sc by facilitating β-sheet formation within this sequence region of PrP. In addition, helical interactions between helix-3 in two or more PrP^C molecules may promote conversion to PrP^Sc.     

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies

The sequential polypeptides (L -Arg-X-Gly)_n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine-rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene-4-sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L -Arg-X-Gly)_n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β-structure. Characteristics of β-turn are excluded from the poly(L -Arg-L -Leu-Gly), which assumed the most pronounced helical conformation. The poly(L -Arg-L -Val-Gly) exerts a significant preference to the β-turn structure compared to that of poly(L -Arg-L -Ala-Gly). Thus the probability for helical, β-structure or β-turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X-residue side chain (β-branching). We concluded that the prepared sequential arginine-containing polypeptides are plausible models for histone fractions, f₃ and f₂α₁.     

Transmembrane Helical Domain of the Cannabinoid CB1 Receptor

Brain cannabinoid (CB₁) receptors are G-protein coupled receptors and belong to the rhodopsin-like subfamily. A homology model of the inactive state of the CB₁ receptor was constructed using the x-ray structure of β₂-adrenergic receptor (β₂AR) as the template. We used 105 ns duration molecular-dynamics simulations of the CB₁ receptor embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer to gain some insight into the structure and function of the CB₁ receptor. As judged from the root mean-square deviations combined with the detailed structural analyses, the helical bundle of the CB₁ receptor appears to be fully converged in 50 ns of the simulation. The results reveal that the helical bundle structure of the CB₁ receptor maintains a topology quite similar to the x-ray structures of G-protein coupled receptors overall. It is also revealed that the CB₁ receptor is stabilized by the formation of extensive, water-mediated H-bond networks, aromatic stacking interactions, and receptor-lipid interactions within the helical core region. It is likely that these interactions, which are often specific to functional motifs, including the S(N)LAxAD, D(E)RY, CWxP, and NPxxY motifs, are the molecular constraints imposed on the inactive state of the CB₁ receptor. It appears that disruption of these specific interactions is necessary to release the molecular constraints to achieve a conformational change of the receptor suitable for G-protein activation.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

Effect of pH, Mg, CO(2) and Mercurials on the Circular Dichroism, Thermal Stability and Light Scattering of Ribulose 1,5-Bisphosphate Carboxylases from Alfalfa, Spinach and Tobacco

Circular dichroism, differential scanning calorimetry and light-scattering measurements of ribulose 1,5-bisphosphate carboxylase (E.C. 4.1.1.39) from alfalfa, spinach and tobacco show: a) The conformation and thermal stability of the native carboxylases are sensitive to changes in pH and to activation of the enzyme with Mg²⁺ and CO₂. The helical content, denaturation temperature (T_d) and specific enthalpy of denaturation (Δq) decreased with increase in pH. Addition of Mg²⁺ and CO₂ at pH 9 increased T_d by 4 to 5 C; at pH 7.5 the changes in T_d were smaller. b) Addition of mercurials produced changes in conformation and thermal stability. The decrease in helical content of the enzymes with increase in pH was enhanced by the addition of p-chloromercuribenzoate. At pH 9, addition of p-chloromercuribenzoate or of 1-(3-(chloromercuri)-2-methoxypropyl)urea decreased T_d by 11.4 to 20.2 C and Δq by 2.1 to 2.8 calories per gram. c) The spinach carboxylase undergoes the largest and the tobacco the smallest changes in conformation and thermal stability upon change in pH or treatment with mercurials. d) The calorimetric data suggest that the large and small subunits are heat denatured independently but at the same temperature. e) Light scattering measurements at pH 9 of p-chloromercuribenzoate treated tobacco enzyme showed that there is no dissociation into subunits upon heating to temperatures greater than T_d. A `ball and string' model for the carboxylase molecule is proposed to reconcile independence of subunit denaturation with apparent strong interactions between subunits.     

Scrutiny of the mechanism of small molecule inhibitor preventing conformational transition of amyloid-β42 monomer: insights from molecular dynamics simulations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by loss of intellectual functioning of brain and memory loss. According to amyloid cascade hypothesis, aggregation of amyloid-β₄₂ (Aβ₄₂) peptide can generate toxic oligomers and their accumulation in the brain is responsible for the onset of AD. In spite of carrying out a large number of experimental studies on inhibition of Aβ₄₂ aggregation by small molecules, the detailed inhibitory mechanism remains elusive. In the present study, comparable molecular dynamics (MD) simulations were performed to elucidate the inhibitory mechanism of a sulfonamide inhibitor C1 (2,5-dichloro-N-(4-piperidinophenyl)-3-thiophenesulfonamide), reported for its in vitro and in vivo anti-aggregation activity against Aβ₄₂. MD simulations reveal that C1 stabilizes native α-helix conformation of Aβ₄₂ by interacting with key residues in the central helix region (13–26) with hydrogen bonds and π–π interactions. C1 lowers the solvent-accessible surface area of the central hydrophobic core (CHC), KLVFF (16–20), that confirms burial of hydrophobic residues leading to the dominance of helical conformation in the CHC region. The binding free energy analysis with MM–PBSA demonstrates that Ala2, Phe4, Tyr10, Gln15, Lys16, Leu17, Val18, Phe19, Phe20, Glu22, and Met35 contribute maximum to binding free energy (?43.1 kcal/mol) between C1 and Aβ₄₂ monomer. Overall, MD simulations reveal that C1 inhibits Aβ₄₂ aggregation by stabilizing native helical conformation and inhibiting the formation of aggregation-prone β-sheet conformation. The present results will shed light on the underlying inhibitory mechanism of small molecules that show potential in vitro anti-aggregation activity against Aβ₄₂.     

Long time molecular dynamic simulation on the agonist binding and activation of the β2-adrenergic receptor

β₂-Adrenergic receptor (β₂AR) plays a critical role in mediating the effects of catecholamine hormones. Due to the flexibility of the structure of its active state, study of agonist–β2AR is usually performed by molecular dynamic (MD) simulation. In this study we show the representative characteristics of agonist binding and activation on β₂AR by MD simulation. The binding site and the conformational changes in the specific regions of β2AR are reasonable which confirmed the conclusion that agonist–β2AR reached its active-like state. We have studied the disruption of non-covalent intramolecular interactions, including the conserved DRY motif, the rotamer toggle switch and the ionic lock, the cytoplasmic ends of transmembranes 5 and 6, and some water-mediated hydrogen bond network regions. We conclude that agonist induced β₂AR to its active conformation, or at least the active-like conformation.     

Molecular dynamics simulations on the first two helices of Vpu from HIV-1

Vpu is an 81 amino acid protein of HIV-1 with two phosphorylation sites. It consists of a short N-terminal end traversing the bilayer and a longer cytoplasmic part. The dual functional role of Vpu is attributed to these topological distinct regions of the protein. The first 52 amino acids of Vpu (HV1H2) have been simulated, which are thought to be embedded in a fully hydrated lipid bilayer and to consist of a transmembrane helix (helix-1) connected via a flexible linker region, including a Glu-Tyr-Arg (EYR) motif, with a second helix (helix-2) residing with its helix long axis on the bilayer surface. Repeated molecular dynamics simulations show that Glu-28 is involved in salt bridge formation with Lys-31 and Arg-34 establishing a kink between the two helices. Helix-2 remains in a helical conformation indicating its stability and function as a "peptide float," separating helix-1 from the rest of the protein. This leads to the conclusion that Vpu consists of three functional modules: helix-1, helix-2, and the remaining residues toward the C-terminal end.     

Nonnative helical motif in a chaperone-bound protein fragment

The effect of cotranslationally active chaperones on the conformation of incomplete protein chains is poorly understood. The secondary structure of a 77-residue chaperone-bound N-terminal protein fragment corresponding to the first five helices (A-E) of apomyoglobin (apoMb_1-77) is investigated here at the residue-specific level by multidimensional NMR. The substrate-binding domain of DnaK, DnaK-β, is employed as a chaperone model. By taking advantage of the improved spectral quality resulting from chaperone deuteration, we find that DnaK-β-bound apoMb_1-77 displays a region of nonnative helicity at residues away from the main chaperone binding site. The nonnative structural motif comprises portions of the native D and E helices and has similar characteristics to the reported nonnative DE helical region of acid-unfolded full-length apoMb. Upon incorporation of the missing C-terminal amino acids, a structural kink develops between residues 56 and 57, and two separate native D and E helices are generated. This work highlights, for the first time to our knowledge, the presence of a nonnative helical motif in a large chaperone-bound protein fragment under physiologically relevant solution conditions.     

Conformational aspects of polypeptides. XXV. Solvent and temperature effects on the conformations of copolymers of benzyl and methyl L-aspartate with nitrobenzyl L-aspartate

A series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate and with β-mcthyl L -aspartatc in helix-supporting and helix-breaking conditions have been reexamined by using ultraviolet isotropic, absorption, optical rotatory dispersion, and circular dichroism techniques. Many different conformations are apparent, depending on solvent and temperature. Chloroform, trifluoroethanol, and methylene dichloride support the left-handed helical conformation of the copolymers containing less than about 20 mole-% nitroaromatic residues and the right-handed helical conformation of the copolymers containing more than approximately 30 mole-% nitroaromatic residues. In trifluoroacetic acid all the copolymers are in a random-coil conformation. In hexa-fluoroacetone trihydrate and in trimethyl phosphate, the copolypeptides with low nitroaromatic residues content are predominantly in a disordered conformation, while those with high nitroaromatic residues content show a right-handed helical array. Reversible helix-ramlom-coil transitions are observed with increasing temperature in trimethyl phosphate. An example of right-handed-left-handed helix reversible transition with temperature is reported in a chloroform-trimethyl phosphate (2:1) mixture. Nitrobenzyl-nilrobenzyl side-chain interactions in chloroform, but not in trifluoroacetic acid or in trimethyl phosphate, have been confirmed. For the first time we report the circular dichroism spectra in which the n-π* peptide band of a left-handed helical conformation is almost completely evident.     

Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β2-adrenergic receptor

The β₂-adrenergic receptor (β₂-AR) agonist [³H]-(R,R′)-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (K_i values) of the stereoisomers of a series of 4′-methoxyfenoterol analogs in which the length of the alkyl substituent at α′ position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [³H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC₅₀ values, were determined in HEK293 cells expressing the β₂-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α′ position. The results also indicate that the K_i values obtained using [³H]-(R,R′)-methoxyfenoterol as the marker ligand modeled the EC₅₀ values obtained from cAMP stimulation better than the data obtained using [³H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β₂-AR conformation probed by [³H]-(R,R′)-4′-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-AR is governed to a greater extend by steric effects than binding to the [³H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

Effect of chain length and concentration of anionic surfactants on the conformational transitions of poly(L-ornithine) and poly(L-lysine) in aqueous solution

The conformation of poly(L-ornithine) (PLO) and poly(L-lysine) (PLL) in solutions of sodium alkyl sulfates, CH₃(CH₂)_nSO₄Na with n = 7, 9, 11, 13 and 15 was studied by circular dichroism. PLO adopts a helical conformation in all 5 homologs and PLL a β-form in only 4 of the homologs. With octyl sulfate PLL has a helical conformation instead. These conformations were observed in solution of surfactants both below and above the critical micelle concentration.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F₁ with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F₁ sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β_DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β_E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.     

Membrane interactions of a self-assembling model peptide that mimics the self-association,structure and toxicity of Aβ(1–40)

β-amyloid peptide (Aβ) is a primary protein component of senile plaques in Alzheimer's disease (AD) and plays an important, but not fully understood role in neurotoxicity. Model peptides with the demonstrated ability to mimic the structural and toxicity behavior of Aβ could provide a means to evaluate the contributions to toxicity that are common to self-associating peptides from many disease states. In this work, we have studied the peptide–membrane interactions of a model β-sheet peptide, P_11-2 (CH₃CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH₂), by fluorescence, infrared spectroscopy, and hydrogen–deuterium exchange. Like Aβ(1–40), the peptide is toxic, and conditions which produce intermediate oligomers show higher toxicity against cells than either monomeric forms or higher aggregates of the peptide. Further, P_11-2 also binds to both zwitterionic (POPC) and negatively charged (POPC:POPG) liposomes, acquires a partial β-sheet conformation in presence of lipid, and is protected against deuterium exchange in the presence of lipids. The results show that a simple rationally designed model β-sheet peptide recapitulates many important features of Aβ peptide structure and function, reinforcing the idea that toxicity arises, at least in part, from a common mode of action on membranes that is independent of specific aspects of the amino acid sequence. Further studies of such well-behaved model peptide systems will facilitate the investigation of the general principles that govern the molecular interactions of aggregation-prone disease-associated peptides with cell and/or membrane surfaces.