Random Coil Peptide Research Articles

Solution Structure and Function of the “Tandem Inactivation Domain” of the Neuronal A-type Potassium Channel Kv1.4

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

Translational diffusion measured by PFG‐NMR on full length and fragments of the Alzheimer Aβ(1–40) peptide. Determination of hydrodynamic radii of random coil peptides of varying length

AbstractPFG‐NMR methods were used to measure the translational diffusion coefficients for the Aβ peptide involved in Alzheimer's disease and also for a series of fragments of this peptide. The peptides ranged from a pentamer to the full length Aβ(1–40). They were studied at 25° C and physiological pH in aqueous solution. The measured diffusion coefficients, including those of known monomeric peptides, were fitted without systematic deviations to a scaling law function of the molecular mass. We concluded that under these conditions Aβ(1–40) is in monomeric form. From the diffusion coefficient data, hydrodynamic radii rH were evaluated for the peptides. When combining our results on non‐ or weakly structured peptides with previously reported results on denatured proteins, we found that the hydrodynamic radii for the combined dataset could be well described by the same scaling law relating them to the molecular weight. The same law would even encompass data on single amino acids and di‐ and tripeptides measured by classical methods. From the above‐mentioned experimental data, scaling law parameters were determined. The relation between the measured hydrodynamic radius (rH) and the molecular weight of the polypeptide chain (Mr) for amino acids, peptides and denatured proteins is rH = 0.27Mr0.50 Å. There is a remarkably good fit to this function for the measured hydrodynamic radii in a large range, almost three orders of magnitude, of molecular weights. The numerical value of the exponent, 0.5, is an indication that these polymers behave as Gaussian chains. Copyright © 2002 John Wiley & Sons, Ltd.

Function of GB1 and GB2 subunits in G protein coupling of GABA(B) receptors.

Many G protein-coupled receptors (GPCRs) have recently been shown to dimerize, and it was suggested that dimerization may be a prerequisite for G protein coupling. gamma-aminobutyric acid type B (GABA(B)) receptors (GPCRs for GABA, a major inhibitory neurotransmitter in the brain) are obligate heterodimers of homologous GB1 and GB2 subunits, neither of which is functional on its own. This feature of GABA(B) receptors allowed us to examine which of the eight intracellular segments of the heterodimeric receptor were important for G protein activation. Replacing any of the three intracellular loops of GB2 with their GB1 counterparts resulted in nonfunctional receptors. The deletion of the complete GB2 C terminus significantly attenuated the receptor function; however, the proximal 36 residues were sufficient for reconstitution of wild type-like receptor activity. In contrast, the GB1 C terminus could be deleted and GB1 intracellular loops replaced with their GB2 or mGluR1 equivalents without affecting the receptor function. In addition, a large portion of the GB1 i2 loop could be replaced with a random coil peptide without any functional consequences. Thus, GB2 intracellular segments are solely responsible for specific coupling of GABA(B) receptors to their physiologic effectors, G(i) and G protein-activated K(+) channels. These findings strongly support a model in which a single GPCR monomer is sufficient for all of the specific G protein contacts.

Transient UV Raman Spectroscopy Finds No Crossing Barrier between the Peptide α-Helix and Fully Random Coil Conformation

Transient UV resonance Raman measurements excited within the amide pi --> pi transitions of a 21 unit alpha-helical peptide has for the first time determined a lower bound for the unfolding rate of the last alpha-helical turn to form a fully random coil peptide. A 3 ns T-jump is generated with 1.9 microm laser pulses, which are absorbed by water. Subsequent 3 ns 204 nm UV pulses excite the amide Raman spectra at delay times between 3 ns and 1 ms, to monitor the peptide conformational evolution. We find approximately 180 ns relaxation times which result in a rate constant of >5 x 10(6) s(-1) for unfolding of the last alpha-helical turn. Our data are inconsistent with slow alpha-helix nuclei melting.

The homologous pairing domain of RecA also mediates the allosteric regulation of DNA binding and ATP hydrolysis: a remarkable concentration of functional residues

Switching between the active (ATP and DNA bound) and inactive conformations of the homologous recombination RecA protein is regulated by ATP hydrolysis. First, we use the homologous pairing domain of RecA derived from its mobile loop L2 to show that the interaction of this random coil peptide with the γ-phosphate of ATP results in a peptide β-conformation similar to that previously shown to be induced by DNA binding. Next, we show that in the whole RecA protein two residues in this L2 domain, Gln194 and Arg196, are catalytic amino acid residues for ATP hydrolysis and functionally resemble the corresponding residues engaged in GTP hydrolysis by two distinct classes of G proteins. Finally, we show that the role of DNA and high salt in the stimulation of the ATPase of RecA is to stabilize this highly mobile region involved in hydrolysis. This is a role similar to that described for RGSs in the activation of the GTPase of heterotrimeric G proteins. Therefore, (i) a prototypical DNA-dependent ATPase and ATP-stimulated DNA-binding protein, RecA, and eukaryotic signaling proteins share common stereochemical regulatory mechanisms; and (ii) in a remarkable example of parsimony, loop L2 is a molecular switch that controls both ATP promoted DNA binding and pairing reactions and DNA stimulated ATP hydrolysis.

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study.

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G.

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.

NMR relaxation measurements of backbone proton exchange in proteins permit a distinction between different mechanisms of exchange

The Linderstrøm-Lang model describes proton exchange as proceeding from an ‘open’ state which is in equilibrium with a ‘closed’ state from which exchange cannot occur. This predicts a biexponential exchange process, although limiting conditions can give rise to monoexponential exchange profiles. When monoexponential exchange profiles are observed, insufficient information is present to define the exchange mechanism. We have directly observed a biexponential exchange process, enabling us to distinguish between alternative amide proton exchange models. Our results are not compatible with the standard local unfolding model, in which units of secondary structure open in a concerted fashion, with each of the newly exposed amide protons exchanging at the same rate which they would in random coil peptides.

NMR relaxation measurements of backbone proton exchange in proteins permit a distinction between different mechanisms of exchange

The Linderstr⊘m-Lang model describes proton exchange as proceeding from an ‘open’ state which is in equilibrium with a ‘closed’ state from which exchange cannot occur. This predicts a biexponential exchange process, although limiting conditions can give rise to monoexponential exchange profiles. When monoexponential exchange profiles are observed, insufficient information is present to define the exchange mechanism. We have directly observed a biexponential exchange process, enabling us to distinguish between alternative amide proton exchange models. Our results are not compatible with the standard local unfolding model, in which units of secondary structure open in a concerted fashion, with each of the newly exposed amide protons exchanging at the same rate which they would in random coil peptides.

Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear 1H?15N NMR spectroscopy

The (15)N relaxation rates of the α-aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional (1)H-(15)N NMR spectroscopy. Measurements of (15)N longitudinal (R(N)(N(z))) and transverse (R(N)(N(x,y))) relaxation rates and the {(1)H} (15)N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the (1)H-(15)N lingitudinal two-spin order, R(NH)(2H (infZ) (supN) N(Z)), two-spin antiphase coherence, R(NH)(2H (infN) (supZ) N(x,y)), and the proton longitudinal relaxation rate, R(H)(H (infN) (supZ) ), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide (15)N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the 'core' regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the 'frayed' C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.

Conformational Analysis of Peptides Corresponding to β-Hairpins and a β-Sheet that Represent the Entire Sequence of the α-Spectrin SH3 Domain

In an attempt to identify potential folding initiation sites for a small, all β-protein domain, we have examined the conformational preferences in aqueous solution of peptides that span the entire length of the α-spectrin SH3 domain, using proton nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. Two of the peptides correspond to β-hairpins (m6 and m8), one to the RT-loop (m4, which can be considered as a distorted β-hairpin), one to a β-hairpin created by joining the N and C-terminal strands viaa small linker (m2) and the fifth one to a three-stranded antiparallel β-sheet composed of β-hairpins m6 and m8 (m68). To estimate the distorting effect of the aromatic side-chains of Trp41 and Trp42 on the CD and NMR spectra of peptides m6, m8 and m68, we have also analyzed a short, ten-residue random-coil peptide containing residues 39 to 44 (mC). The CD and NMR results indicate that none of the peptides populates to a large extent a particular secondary structure conformation. However, careful analysis of the NMR data reveals that peptides m6, m8 and m68 could adopt, to a small extent, native-like conformations, although in the case of peptide m68 there is also evidence of the presence of non-native helical conformations. Addition of 30% (v/v) 2,2,2-trifluoroethanol stabilizes the appearance of non-native helical populations in some small regions of peptides m2, m4, m8 and m68, while it induces a native-like conformation in peptide m6. Those fragments corresponding to the two real β-hairpins in the protein are the ones which exhibit some tendency to populate native-like structures (m6 and m8), while the ones corresponding to the long RT-loop (m4) or the newly created one (m2) are mainly unstructured in water solution. Although there could be some local interactions that favor the acquisition of a native secondary structure in this domain, tertiary interactions should play a major role in defining its native secondary structure.

Comparison between the φ Distribution of the Amino Acids in the Protein Database and NMR Data Indicates that Amino Acids have Various φ Propensities in the Random Coil Conformation

It has been indicated that amino acids have various intrinsic φ and ψ propensities, as demonstrated from the comparison between experimental secondary structure propensities and their relative statistical distribution in the protein database for the appropriate region of the Ramachandran plot. However, this does not eliminate the possibility that these experimental propensities are the result of context effects due to the secondary structure environment of the mutated position. To demonstrate that there are at least real intrinsic φ propensities, independent of context effects, we have used two different nuclear magnetic resonance parameters related to the φ dihedral angle ( J 3 αHNcoupling constants and the chemical shift of the C αH proton), determined in random-coil tetra- and pentapeptides, and/or in proteins. Comparison of the experimentally determined values for these parameters with the theoretical ones determined from the analysis by different empirical and theoretical equations of the φ dihedral angle statistical distribution of the amino acids in the protein database, supports the idea that each amino acid has, at least, different φ intrinsic propensities. Consideration of all conformations, or only coil conformations, in the protein database produces similar results. The reasonable correlation between these experimental and theoretical data and the hydrogen- exchange data in random-coil peptides suggests that maximisation of hydrophobic surface-buried and hydrogen-bond formation with the solvent could be responsible for these different random-coil conformational preferences. Analysis of the intrinsic propensities for β-strand, α-helix and polyproline II dihedral angles of the 20 amino acids in coil conformations, indicates that the side-chain of the amino acids is mainly determining the relative preferences for the φ angle.

A single point chiral inversion that selforganizes a randomcoil peptide. Apolar solvent conformation of Boc-( L\\ D)-Glu-Ala-Leu-LysNHMe

The tetrapeptide Boc- L-Glu-Ala-Leu-LysNHMe ( 1) reveals a random coil conformation, based on its Glu(γ) and Lys(ɛ) methylene proton aniosotropic shift, GluNH chemical shift, NOEs in chloroform-DMSO (6:1), and its amide proton temperature coefficients in DMSO, while on similar considerations, the diastereomer Boc- D-Glu-Ala-Leu-LysNHMe ( 2) is characterized as a highly ordered 3/10 type distorted protohelix with a remarkably stable intramolecular salt bridge under these solvent conditions.

Solution dynamics of the trp repressor: A study of amide proton exchange by T1 relaxation

The amide proton exchange rates of Escherichia coli trp repressor have been measured through their effects on the longitudinal relaxation rates of the amide protons. Three types of exchange regimes have been observed: (1) slow exchange (on a minute/hour time-scale), measurable by isotope exchange, but not by relaxation techniques in the core of the molecule; (2) relatively rapid exchange, with the rates on a T1 relaxation time-scale (seconds) in the DNA-binding region and (3) very fast exchange at the N and C termini. The results have been analyzed in terms of the two-site exchange model originally proposed by Linderstrøm-Lang, and of a three-site extension of the model. The values of the intrinsic exchange rates calculated using the two-state model agree with the values expected from the studies of Englander and co-workers for the very fast case of the chain terminals, but disagree with the literature values by two orders of magnitude in the intermediate case found in the DNA-binding region. The implication of these findings is that the "open" state of the two-state model in the DNA-binding region is not completely open and has an intrinsic exchange rate different from that of a random coil peptide. Alternatively, if the literature values of the intrinsic exchange rates are assumed to apply to the open states in all parts of the repressor molecule, two "closed" helical states have to be postulated, in slow exchange with each other, with only one of them in rapid exchange with the open state and hence with the solvent. Kinetically, the two models are indistinguishable.

1H-NMR Studies of the natriuretic peptide urodilatin: sequence-specific resonance assignment

The recently discovered 32 amino-acid natriuretic peptide urodilatin was chemically synthesized and subjected to two-dimensional proton nuclear magnetic resonance (NMR) spectroscopy studies in aqueous solution in order to determine the structural state of urodilatin. In contrast to earlier studies on very closely related peptides, such as cardiodilatin (CDD/ANP-99-126) and brain natriuretic peptide (BNP), spectra of urodilatin were extremely well resolved even in millimolar concentration in H 2O so that the complete sequence specific resonance assignments could be achieved. No long range NOEs could be detected, except between residues close to the single cystine bond. This leads to the conclusion that urodilatin in aqueous solution is a random coil peptide with the exception of the region around the cystine bond

Unfolding of the molten globule state of alpha-lactalbumin studied by 1H NMR.

The urea-induced unfolding of the molten globule state of bovine alpha-lactalbumin was investigated by 1H nuclear magnetic resonance. In the molten globule state, most of the aromatic resonances deviate from their random coil values, indicating that aromatic side chains form some ordered structures in the molten globule state. When the urea concentration increases, the resonances are shifted, and the deviations from the random coil values are diminished. Because the chemical shifts of several random coil peptides are found to be independent of urea concentration, the urea-induced shifts of the resonances in the molten globule state reflect the unfolding transition of some ordered structures. The unfolding transitions measured by individual aromatic resonances do not coincide with each other. The unfolding transition curves obtained from some aromatic resonances are also different from those of the secondary structures measured by circular dichroism spectra. These results clearly show that the unfolding of the molten globule state of alpha-lactalbumin is not a cooperative two-state process.

Dissection of an enzyme by protein engineering: The N and C-terminal fragments of barnase form a native-like complex with restored enzymic activity

A method is described for producing fragments of a protein suitable for studies of protein folding. The codon for a single methionine residue is introduced into the cloned gene of barnase, and the gene product cleaved with cyanogen bromide. The site of mutation was chosen to be at the surface of the protein in a region connecting segments of secondary structure in the native enzyme. The α + β protein was mutated from Val36 → Met, and split into two fragments, B(1–36) containing the α-helical regions and B(37–110), the β-sheet. The fragments were purified by ion exchange chromatography. Neither retains catalytic activity. Fluorescence, circular dichroism, and 1H nuclear magnetic resonance data indicate that their structures are each close to that of random-coil peptides. The two fragments associate to form a tight complex ( K d = 0.2 to 0.6 μm), which displays spectroscopic properties similar to those of the uncleaved protein. The catalytic activity is restored in the complex with a value for K m similar to that for native enzyme but with k cat reduced about three- to fourfold. The second-order rate constant for association on mixing fragments in the concentration range 2.5 to 7.5 μ m is 1 × 10 5 s −1 m −1 .

Experimental and computational infrared CD studies of prototypical peptide conformations

The infrared vibrational circular dichroism (VCD) spectral features of prototypical peptide secondary structures were reported previously by Yasui and Keiderling [Yasui, S.C., & Keiderling, T.A. (1986) Biopolymers 25, 5]. These results demonstrated that the "random coil" peptide conformation exhibits VCD signals which are approximately mirror-image features of those exhibited by alpha-helical conformers. We report here a comparison of observed VCD spectra with those computed for several secondary structures, using the extended coupled oscillator formalism employed previously to compute VCD spectra of model DNA [Zhong et al. (1990) Biochemistry 29, 7485]. These studies suggest that the so-called random-coil peptide conformation has distinct short-range order and appears to be a left-handed, helical structure.

Immunogenicity of peptides having pre-determined alpha-helical and alpha-alpha fold topologies

A panel of three synthetic peptides based on the 310–327 region of mouse LDH-C4 was used to examine the effect of peptide conformation on immunogenicity. The peptides, without prior conjugation to carrier molecules, were injected into outbred mice and the antisera were assayed for peptide- and LDH-C 4-reactive antibodies by ELISA. An 18-residue random coil peptide (α N) and an 18-residue amphipathic alpha-helix peptide (α 1) were weakly immunogenic. A conformationally stable 40-residue alpha-alpha fold peptide (α 3) was highly immunogenic. The antibodies elicited by α 3 reacted strongly with the native molecule by ELISA. Solution-phase binding assays were used to further characterize the specificity of the sera from two mice immunized with α 3 Antibodies from one of the mice appeared to recognize the helical portion of the peptides, while antibodies from the other mouse reacted only with the immunogen and may be specific for the non-natural β bend residues or possibly a topographic determinant peculiar to the anti-parallel helices. Serum from neither mouse was able to recognize the native molecule in solution. Peptides intended to mimic topographic determinants for the purpose of synthetic vaccine development may have to be more complex than those used in this study in order to induce high-affinity antibodies capable of exerting a significant biological effect.

Polypeptide dynamics: Experimental tests of an optimized Rouse–Zimm type model

A theory for long time random coil peptide dynamics is developed based on a generalization of the optimized Rouse–Zimm model of Perico et al. [J. Chem. Phys. 87, 3677 (1987)] and Perico [J. Chem. Phys. 88, 3996 (1988) and Biopolymers 28, 1527 (1989)]. The generalized model employs the rotational potential energy for specific amino acid residues and amino acid friction coefficients to compute all input parameters in the model. Calculations of the fluorescence depolarization correlation function P2(t ) and of the local persistence length are found to be sensitive to the amino acid sequence, the length of the polypeptide chain, and the location of the probe. Model computations of P2(t ) are compared with new experimentally determined rotational correlation times (of the order of nanoseconds) from fluorescence depolarization measurements of three different synthetic 17-residue peptides, each containing a single tryptophan (TRP) residue as a probe. In addition, the previous anisotropy measurements on ACTH, glucagon, and their fragments are discussed and compared with the model calculations. Our results indicate that the theory gives a reasonable prediction for the fluorescence depolarization correlation times of random coil polypeptides, but the calculated rotational correlation function predicts a much faster initial decay and a slower final decay than is observed. Possible theoretical improvements are discussed.