Preferred side‐chain conformation of arginine residues in a triple‐helical structure

The crystal structure of the triple‐helical peptide (Pro‐Hyp‐Gly)₃‐Pro‐Arg‐Gly‐(Pro‐Hyp‐Gly)₄ (POG3‐PRG‐POG4) was determined at 1.45 Å resolution. POG3‐PRG‐POG4 was designed to permit investigation of the side‐chain conformation of the Arg residues in a triple‐helical structure. Because of the alternative structure of one of three Arg residues, four side‐chain conformations were observed in an asymmetric unit. Among them, three adopt a ttg^?t conformation and the other adopts a tg^?g^?t conformation. A statistical analysis of 80 Arg residues in various triple‐helical peptides showed that, unlike those in globular proteins, they preferentially adopt a tt conformation for χ₁ and χ₂, as observed in POG3‐PRG‐POG4. This conformation permits van der Waals contacts between the side‐chain atoms of Arg and the main‐chain atoms of the adjacent strand in the same molecule. Unlike many other host–guest peptides, in which there is a significant difference between the helical twists in the guest and the host peptides, POG3‐PRG‐POG4 shows a marked difference between the helical twists in the N‐terminal peptide and those in the C‐terminal peptide, separated near the Arg residue. This suggested that the unique side‐chain conformation of the Arg residue affects not only the conformation of the guest peptide, but also the conformation of the peptide away from the Arg residue. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1000–1009, 2014.     

Structural and functional role of leucine residues in proteins

Circular dichroism and potentiometric titration studies of leucine random copolymers in aqueous solutions, as well as a comparison of the conformational stability in poly-α-amino acids, indicate that leucine may possibly be the amino acid with the highest propensity for forming α-helical structures. This suggests that leucine might be found most frequently in the helical regions of proteins. A survey was made on 15 different proteins containing 2473 residues with known sequence and conformation determined by X-ray crystallography: carboxy-peptidase A, α-chymotrypsin, cytochrome b₅, elastase, ferricytochrome c, α- and β-hemoglobin, insulin, lysozyme, myogen, myoglobin, papain, ribonuclease A, staphylococcal nuclease, and subtilisin BPN′. It was found that 888 residues in these proteins are in helices, and 422 of them reside in the internal turns of helical regions. While Glu, Ala, Leu and His were found to be present with the highest percentages in helical regions, Leu was clearly the most abundant residue in the inner helical cores of proteins. Polar residues are found preferentially at the helix-coil boundary regions; Asp and Glu at the N-terminal and His, Lys and Arg at the C-terminal helical ends. These findings agree with Ptitsyn's (1969) analysis on seven proteins containing 1132 residues. A more comprehensive analysis in the present survey showed that Ile, Met and Val occur with the greatest frequency in the β-regions of proteins. Leu was also found as the strongest structure-forming residue in proteins (total helical and β-regions). The functional-structural role of leucine was established by showing that it occurs most frequently among residues surrounding the heme in five of the heme proteins. In addition, the greater abundance of leucine as neighbors to active-site residues in enzymes provides strong evidence that hydrophobic residues create a non-aqueous environment, aiding the polar residues in substrate binding and enzymic catalysis. Examples of conservative and non-conservative mutations of leucine in heme proteins are given to illustrate the structure—function relation of proteins, and explain why most leucine residues in the insulin, hemoglobin, and cytochrome c homologs are invariant. Finally, the strong helical-forming power of leucine, as demonstrated experimentally in synthetic copolypeptides and its high occurrence in the inner helical cores of proteins, suggests that it could have a major role as nucleation centers in the folding and evolution of large protein molecules.     

Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box”

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as “capping boxes,” we have reexamined the side chain–main chain interactions in a varient sequence using ¹H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved: Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant. Thus the capping box sequence Ser-Glu-Asp-Glu inhibits N terminal fraying of the N terminus of alpha helix in these peptides, and shows the side chain–main chain interactions proposed by Harper and Rose. It thus acts as a helix initiating signal. Since normal a helix cannot propagate beyond the N terminus of this structure, the box acts as a termination signal in this direction as well. © 1994 John Wiley & Sons, Inc.     

Salt-induced conformational change of random copolymers (Lys50Tyr50)n and (Lys50Phe50)n studied by 1H- and 13C-nuclear magnetic resonances. Aggregation behavior of helical segments

¹H- and ¹³C-nmr studies of conformational transitions of random amino acid copolymers containing aromatic residues (Lys⁵⁰Tyr⁵⁰)_n and (Lys⁵⁰Phe⁵⁰)_n in the presence of neutral salts were performed to serve as models of the aggregation behavior of polypeptides of biological significance. The ¹H and ¹³C signal intensities of Tyr and Phe residues decreased preferentially with increasing concentration of neutral salts such as NaCl and NaClO₄. This behavior contrasts with that of (Lys)_n in the presence of similar neutral salts, where the displacement of the ¹³C signal is clearly seen on transition from the random-coil to the helical conformation. On the basis of the previous conformational studies, the loss of the peak areas is ascribed to the presence of immobilized helical segments by hydrophobic interaction between aromatic side chains. The remaining resonances are due to the residual random-coil regions, since the values of nuclear Overhauser enhancements and chemical shifts are unchanged in the presence and absence of the neutral salts.     

The conformation of an alamethicin in methanol by multinuclear NMR spetroscopy and distance geometry/simulated annealing

The solution conformation of the antibiotic peptide alamethicin was investigated using multi-nuclear spectroscopy and the distance geometry/simulated annealing algorithms from the program DSPACE. ¹H-, ¹³C-, and ¹⁵N-nmr chemical shifts and homonuclear ¹H coupling constants suggest that the molecule is flexible in the vicinity of Gly-11 and Leu-12. The temperature dependence of the amide proton chemical shifts indicates that there is flexibility in the middle of the 20 residue peptide and provides evidence that, at the very N-terminus, the molecule adopts a 3₁₀-helical conformation. The large differences in the ¹³C chemical shifts of the pro-R and pro-S methyls of the α-aminoisobutyric acid residues were used to constrain those residues to the right-handed helical conformation in the distance geometry/simulated annealing algorithms. A family of 24 structures was generated but did not converge to a common conformation when superimposed over the entire polypeptide sequence. The molecules did converge to a helical conformation over residues 1–10 and residues 13–18. The lack of convergence when the entire lengths of the molecules are superimposed is explained by the flexibility of the peptide near Gly-11/Leu-12. The results suggest that the protein consists of two helices connected by a flexible “hinge.” The flexibility of the molecule is discussed with respect to the macrodipole model of voltage gating. © 1995 John Wiley & Sons, Inc.     

High-resolution structural profile of hylaseptin-4: Aggregation,membrane topology and pH dependence of overall membrane binding process

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH₂) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d₃₈ micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.     

Omega amino acids in peptide design: incorporation into helices

Incorporation of easily available achiral ω-amino acid residues into an oligopeptide results in substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic. The central Gly-Gly segment of the helical octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-Ome(1) has been replaced by δ-amino-valeric acid (δ-Ava) residue in the newly designed peptide Boc-Leu-Aib-Val-δ-Ava-Leu-Aib-Val-OMe(2). ¹H-nmr results clearly suggest that in the apolar solvent CDCl₃, the δ-Ava residue is accommodated into a folded helical conformation, stabilized by successive hydrogen bonds involving the NH groups of Val(3), δ-Ava(4), and Leu(5). The δ-Ava residue must adopt a gauche-gauche-trans-gauche-gauche conformation along the central polymethylene unit of the aliphatic segment, a feature seen in an energy-minimized model conformation based on nmr parameters. The absence of hydrogen bonding functionalities, however, limits the elongation of the helix. In fact, in CDCl₃, the folded conformation consists of an N-terminal helix spanning residues 1–4, followed by a Type II β-turn at residues 5 and 6, whereas in strongly solvating media like (CD₃)₂SO, the unfolding of the N-terminal helix results in β-turn conformations at Leu(1)-Aib(2). The Type II β-turn at the Leu(5)-Aib(6) segment remains intact even in (CD₃)₂SO. CD comparisons of peptides 1 and 2 reveal a “nonhelical” spectrum for 2 in 2,2,2-trifluoroethanol. © 1996 John Wiley & Sons, Inc.     

Regular left-handed fragment of amylose: crystal and molecular structure of methyl-α-maltotrioside, 4H2O

The crystal structure of methyl-α-maltotrioside tetrahydrate C₁₆H₃₄O₁₆, 4H₂O), has been established by direct methods from 2269 independent reflections and refined to a final R value of 0.054. The crystal belongs to the orthorhombic system, space group P2₁2₁2₁ and has a unit cell of dimensions: a = 1.037 (1), b = 2.439 (1) and c = 1.065 (1) nm. The three glucose residues have the ⁴C₁ pyranose conformation and are α-(1–4)-linked. The conformation of the glycosidic linkage is characterized by torsion angles (φ, ψ) which take the values (82.2, −148.9) between the non-reducing and the middle residue and (82.8, −151.8) between the middle residue and the reducing one. The primary hydroxyl groups exist in a gauche-gauche conformation. This structure is also characterized by the lack of intramolecular hydrogen bonding between secondary hydroxyl groups belonging to contiguous residues. The molecules are held together by a complicated network of hydrogen bonds involving all the hydroxylic groups and the water molecules. the three dimensional arrangement corresponds to a regular alternation of antiparallel bilayers strongly linked by water molecules. A survey of the distribution of the glycosidic torsion angles in all known linear α-(1–4)-linked d-glucose residues, discloses the existence of three stable conformers. This crystal structure provides the first experimental evidence of a regular left-handed fragment of the amylosic chain in a highly hydrated neighbourhood. Furthermore, the helical conformation adopted by the trisaccharide gives rise to helical parameters which are close to those found experimentally for native A and B amyloses. The relevance of the present results to the rationalization of the polymorphic transformation of amylose, along with its crystallization habits is also discussed.     

Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Insight into a Random Coil Conformation and an Isolated Helix: Structural and Dynamical Characterisation of the C-Helix Peptide from Hen Lysozyme

A 17 residue peptide corresponding to the C-helix of hen lysozyme (residues 86 to 102) has been investigated in detail to assess the factors that determine its conformation in both aqueous and trifluoroethanol (TFE) solutions. A thorough characterisation of the peptide by CD and NMR techniques under both conditions has been performed including the determination of complete NMR proton sequential assignments, and measurement of NOE effects,³J_HNαcoupling constants, temperature coefficients and residue-specific hydrogen-exchange rates. In water, the peptide adopts a largely unstructured conformation and NMR data, particularly coupling constants and chemical shift deviations, have been shown to agree closely with predictions from a model for a random coil based on the φ,ψ distributions in a protein database. This indicates that under these conditions the intrinsic conformational preferences of the individual amino acid residues are the dominating factors that determine the population of conformers adopted. With increasing concentrations of TFE a cooperative transition to an extensively helical conformation occurs and the resultant changes in CαH chemical shifts have been shown to correlate with the changes in φ,ψ populations. Using NOE and coupling constant data for this state, an ensemble of structures has been calculated and provides a model for a helix in the absence of tertiary interactions. In this model fluctuations, which increase in amplitude towards the termini, occur about the average helical φ,ψ angles and are responsible for increasing the values of³J_HNαcoupling constants above those anticipated for a static helix. The residue-specific rates of hydrogen exchange for the peptide in 50% TFE-d₃are consistent with such a model, the maximum protection from exchange being observed for residues in the centre of the helix.     

Synthetic α-helical peptides incorporating intercalators for DNA recognition

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz ¹H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential d_NN(i, i+1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an α-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.     

The effect of a terminal chiral residue on the helicity of a peptide chain

Summary We studied the effect of incorporating a chiral terminal amino acid residue (L-leucine) on the helical screw sense of a previously characterized achiral helical polypeptide module-[glycine-(C^α,α-di-n-butylglycine)-glycine]₂-by means of CD spectroscopy and conclude that the presence of this residue at the carboxyl terminal induces a predominantly left handed helical conformation of the helix.     

Modified chemotactic peptides: Synthesis,conformation, and activity of HCO-Thp-Ac6c-Phe-OMe

HCO-Thp-Ac₆c-Phe-OMe (3) has been synthesized as a new analogue of the prototypical chemotactic agent HCO-Met-Leu-Phe-OMe (fMLP-OMe). Compound 3 contains 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 1-aminocyclohexane-1-carboxylic acid (Ac₆c) as achiral, conformationally restricted mimics of Met and Leu, respectively. In the crystal, the formyltripeptide adopts an helical conformation at the Thp and Ac₆c residues, of the type α_R and α_L, respectively, whereas the C-terminal phenylalanine is quasi-extended. A system of two consecutive γ-turns, centered at the first two residues, better explains the nmr data as compared with an alternative β-turn structure. The conformation of the new analogue 3 is compared with those of two related peptides containing Thp as N-terminal residue. The biological activity of 3 has been determined on human neutrophils and compared to that of the previously studied model [Ac₆c²] fMLP-OMe. While the above analogue is highly active in the superoxide anion production, the new tripeptide 3 is practically unable to elicit any of the tested biological activities. © 1996 John Wiley & Sons, Inc.     

Conformation of thymosin β9 in water/fluoroalcohol solution determined by NMR spectroscopy

The conformation of thymosin β₉ in solution of 40% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol-d₂ in water has been investigated by two-dimensional ¹H-nmr spectroscopy. Under this condition thymosin β₉ adopts an ordered structure. The determination of the conformation of the peptide was based on a set of 304 approximate interproton distance constraints derived from nuclear Overhauser enhancement measurements. The conformation of thymosin β₉ includes two helical regions from residues 4 to 27 and 32 to 41. The two helices are separated by a poorly defined loop region between amino acids 28 and 31; the N-terminus of thymosin B₉ shows random-coil structure only. © 1997 John Wiley & Sons, Inc. Biopoly 41: 623–634, 1997     

Helical screw sense of peptide molecules: The pentapeptide system (Aib)4/L-Val[L-(αMe)Val] in the crystal state

The crystal-state preferred conformations of six N^α-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid (Aib) residues followed by one chiral L -valine (L -Val) or C^α-methyl-L -valine [(αMe)Val] residue at the C-terminus, have been assessed by x-ray diffraction analysis. In all of the compounds the  (Aib)₄ sequence is folded in a regular 3₁₀-helical conformation. In the four pentapeptides characterized by the L -(αMe)Val residue two conformationally distinct molecules occur in the asymmetric unit. Conversely, only one molecule is observed in the asymmetric unit of two pentapeptides with the C-terminal L -Val residue. In the L -Val based peptides the helical screw sense of the  (Aib)₄ sequence is right-handed, whereas in the L  (αMe)Val analogues both right- and left-handed helical screw senses concomitantly occur in the two crystallographically independent molecules. © 1998 John Wiley & Sons, Inc. Biopoly 46: 433–443, 1998     

Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: Implications in cell selective lysis by d-amino acid containing antimicrobial peptides

Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-^dH containing d-amino acids [^dV5, ^dV8, ^dL11 and ^dK16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H-D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-^dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-^dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-^dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D₂O demonstrated a large difference in dynamics between Mel-H and Mel-^dH, whereby almost all backbone protons of Mel-^dH showed a much faster rate of exchange as compared to Mel-H. Proton T₁ relaxation had suggested a mobile backbone of Mel-^dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-^dH is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.     

1H and 13C nmr studies of cyclic and linear dipeptides containing threonyl,seryl, aspartyl,and histidyl residues

The side-chain conformations of D - orL - Thr, D - or L -Ser, L -Asp, and L - His residues in cyclic and linear dipeptides in D₂O or in DMSO-d₆ are deduced from vicinal (¹H,¹H) and (¹³C, ¹H) coupling constants. Vicinal (¹³C, ¹³C) coupling constants strongly depend on substituents and cannot be used without a more sound analysis. In cyclic dipeptides, the Thr and Ser side chains are folded above the DKP ring, with χ¹ near 60°. The L -Asp side chain interacts more specifically with peptide bonds (χ¹ near 300°). The L - His side chain is more flexible and its conformation depends on the proximity of a second side chain and on solute-solvent interactions. In all cases, this side chain is not completely folded. In linear dipeptides, the conformation of a C-terminal L -His residue is mainly influenced by the end carboxylic group. On the other hand, a N-terminal L -His residue interacts more easily with a neighboring L -Asp residue. In aqueous solution, the imidazole pK_a depends on the proximity of terminal and lateral charged groups but does not reveal any specific interaction in cyclic dipeptides. A comparison between the conformations of cyclic peptides observed in solution, in the crystalline state and calculated by empirical methods, allows one to point out the discriminating role of the packing in crystals, and of solute-solvent interactions in solution.     

Crystal structure of the collagen model peptide (Pro‐Pro‐Gly)4‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)4 at 1.0 Å resolution

The single‐crystal structure of the collagen‐like peptide (Pro‐Pro‐Gly)₄‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)₄, was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple‐helical structure and that its conformation is very similar to that of (Gly‐Pro‐Hyp)₉, which has the typical repeating sequence in collagen. High‐resolution studies on other collagen‐like peptides have shown that imino acid‐rich sequences preferentially adopt a 7/2 triple‐helical structure (θ = 51.4°), whereas imino acid‐lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly‐Hyp‐Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro‐Pro‐Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2‐helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N‐terminus of an adjacent molecule, causing axial displacement, reminiscent of the D‐staggered structure in fibrous collagens. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 436–447, 2013.     

Logical analysis of the mechanism of protein folding III. Prediction of the strong long-range interactions.

It has been found that strong long-range interactions occur in regions having large β-structural potentials. As has been described previously (Nagano, 1974), interactions among regions having both helical and β-structural potentials (αβ-gaβ interactions) are also very important. Accordingly, an idea is presented in this paper that the relative stability of a protein conformation could be estimated by a relatively simple mathematical function of sequence and conformation. The function P(p,q) is called the non-energy part of pseudo-free energy, because minimization of the sum of P(p,q) and energy functions (cf. Levitt, 1974; Warme &; Scheraga, 1974) can be expected to lead to a plausible model of a protein. A merit of the function is that it can help us decide which way to go in manipulating a temporarily built model, e.g. towards a helix-rich protein or towards a β-structure-rich protein. The estimation of P(p,q) as an artificial potential does not use much computer time because only the co-ordinates of the β-carbon atoms (α-carbon atoms if the residue is Gly) are used. It is composed of terms of the long-range interactions P_L and short-range interactions P_S. The term P_L represents the relative strength of helix-helix interactions, helix-β-candidate interactions and β-candidate-β-candidate interactions. It is assumed that both helical and β-structural potentials can be measured as the differences between the predicted function for helix and β-structure, respectively, as defined previously (Nagano, 1973), and the corresponding largest values ever found. A hypothesis that two residues distantly separated in the primary sequence contribute less to the stability of the whole molecule is finally discarded because the true conformation of concanavalin A becomes very unstable compared with its false conformation folded like the main part of subtilisin. The parameters thus determined indicate that the helix-β-candidate interactions are almost as important as the β-candidate-β-candidate interactions. Both helix and loop prediction functions are combined to give the short-range interactions term, P_S, according to whether the region is really helical or not, and to whether it is really looped or not. The function P(p,q) can be used as a criterion for judging whether the predicted conformation is realistic or false, because the parameters can be adjusted to give, within limits, reasonable values of −10 kcal/residue for true conformations and higher than −5 kcal/residue for false conformations.As an application of the present theory of protein folding, the tertiary structure of bacteriophage T4 lysozyme is predicted and presented in Figure 1, prior to the X-ray structure becoming available.     

Analysis of N-terminal capping using carbonyl-carbon chemical shift measurements

The model peptide XAAAAEAAARAAAARamide is used to examine the contributions of an N-terminal capping interaction to the conformation and stability of a helical ensemble. The reference peptide has an alanine residue at position X while the capping peptide has a serine residue at this position. The helical ensemble was characterized using circular dichroism measurements and carbonyl-carbon chemical shift measurements of selectively enriched residues. The distribution of helicity within the ensemble of the reference peptide at pH 11 and 0°C appears symmetrical, having a uniform central helix and frayed ends. This distribution is truncated at pH 6 by the repulsive electrostatic interaction between the positively charged α-amino group and the positively charged end of the helical macrodipole. The capping peptide forms a side-chain/main-chain hydrogen bond involving the serine residue and amide of alanine 4. The presence of this hydrogen bond generates a unique motif in the chemical shift profile of its helical ensemble. The conformational stabilization contributed by this hydrogen bond, although cooperatively distributed throughout the helical ensemble, is preferentially focused within the first helical turn. The stabilization provided by this hydrogen bond is able to offset the truncation of the helical ensemble generated by the repulsive electrostatic interaction observed at pH 6. Proteins 33:167–176, 1998. © 1998 Wiley-Liss, Inc.