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.     

Molecular states in single-stranded adenylate chains by relaxation analysis

The single-strand helix-coil transition in various oligo- and polyadenylates is characterized by means of an improved cable temperature-jump technique. In all the polymers studied {poly(rA), poly(dA), poly[A(m^2′)] and poly[A(e^2′)]} helix-coil relaxation is observed in the time range from 30 to 1000 nsec. Relaxation-time constants observed at wavelengths λ<280 nm (τ_α) are different from those found at λ >280 nm (τβ), indicating the presence of more than two conformational states. The time constants τ_α increase in the series poly(dA), poly[A(m^2′)], constants τ_β/τ_α is approximately 2.5, except in poly(dA) where τ_β/τ_α ≈ 9. Relaxation measurements with r(A)_n- oligomers show a decrease in conformational mobility with increasing chain length. The relaxation curves also demonstrate that “internal” residues have lower reaction rates than residues at the ends of the oligomer chain. Measurement in D₂O reveal a solvent isotope effect for τ_α of +87% for poly(rA), and of +53% for poly(dA), whereas no isotope effect is found in τ_β. The absence of “slow” relaxation processes in the model compound 9,9′ -trimethylenebisadenine shows that the relatively low rate of the single-strand helix-coil transitions is due to the coupling of base stacking with the folding of the sugar–phosphate chain. The absence of a seprate relaxation process (corresponding to τ_β) in 9,9′-trimethylenebisadenine, as well as in the dinucleotides ApC and CpA, suggests that this relaxation process is dependent upon the presence of both the sugar–phosphate chain and of adjacent adenine bases. The experimental data provide evidence that there is more than one ordered conformation in various single-stranded oligo- and polyadenylates and that the transition between these conformations is influenced by the sugar conformation.     

Characterization of the dynamic behavior of r(ACC) and r(AAC) with NMR relaxation data and both metropolis monte carlo and molecular dynamics simulations

The solution-state behavior of two triribonucleotides, adenylyl(3′-5′) adenylyl (3′-5′) cytidine [r(AAC)] and adenylyl (3′-5′) cytidylyl (3′-5′) cytidine [r(ACC)], was studied with spectroscopic and molecular modeling methods. Melting temperatures of 299 and 294 K for r(AAC) and r(ACC), respectively, were obtained from ultraviolet absorption (UV) and circular dichroism (CD) temperature profiles of the order-disorder transition. The behavior of the Raman marker modes is consistent with greater stability of r(AAC) compared to that of r(ACC). Nuclear magnetic resonance (nmr) relaxation data (homonuclear cross-relaxation rates, proton selective and nonselective longitudinal relaxation times, and carbon longitudinal relaxation times) were measured at 283, 296, and 318 K for both trimers. In parallel, the major types of conformations were explored with Metropolis Monte Carlo (MMC) and molecular dynamics (MD) simulations to obtain representations of both slow and fast events. Fitting of experimental data showed that although the MMC conformations do not represent an exhaustive list of conformers in solution, the canonical helical form (A-RNA type) should coexist at low temperature with significant populations of other less classical conformers such as half-stacked (HS), bulged (BU), and reverse-stacked (RS). Fitting of the experimental relaxation data ensemble at 283 K led to very different representations for the two trimers. r(AAC) was shown to have a fairly compact, rigid structure (angular order parameter, S²_ang ∼ 0.9, correlation time for internal motion, τ_e ∼ 0.1 ns), which undergoes fairly rapid overall tumbling characterized by the correlation time τ_c ∼ 0.6 ns, whereas r(ACC) exhibits much more flexibility (S²_ang ∼ 0.7, τ_e ∼ 0.1 ns) and slower molecular reorientation (τ_c ∼ 1.0 ns). The values of S²_ang tended to confirm that large amplitude fluctuations did not occur on the relaxation timescale (ns). In the course of this paper, a widely accepted concept was shown to be questionable. As regards the nmr relaxation data, simulations show that for fairly small nucleic acids (τ_c < 2.0 ns) the second term of the model-free spectral densities is not negligible for representative motional models (S²_ang values < 0.9 and τ_e values in the 0.05–0.2 ns range). The difference in the dynamic behavior of r(AAC) and r(ACC) can be explained by the greater propensity of the A-A sequence to stack as compared to that of A-C. © 1996 John Wiley & Sons, Inc.     

A comparison of 15N NMR relaxation measurements with a molecular dynamics simulation: Backbone dynamics of the glucocorticoid receptor DNA-binding domain

The rapid motions of the backbone of the DNA-binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton-detected heteronuclear NMR experiments on ¹⁵N-labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S²) and an effective correlation time (τ_e) for the internal motion of each amide bond. A back calculation, using the same model, yielded the {¹H}-¹⁵N nuclear Overhauser effects (NOEs) and the ¹⁵N spin-lattice relaxation times (T₁) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major α-helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S²-values was as good as quantitative for most of the residues, except for some residues that were subject to a more large-scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile-487 between the charged oxygens of the side chain of Asp-485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental τ_e-values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (<0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower τ_e-values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile-487. © 1993 Wiley-Liss, Inc.     

Femtosecond Carotenoid to Retinal Energy Transfer in Xanthorhodopsin

Xanthorhodopsin of the extremely halophilic bacterium Salinibacter ruber represents a novel antenna system. It consists of a carbonyl carotenoid, salinixanthin, bound to a retinal protein that serves as a light-driven transmembrane proton pump similar to bacteriorhodopsin of archaea. Here we apply the femtosecond transient absorption technique to reveal the excited-state dynamics of salinixanthin both in solution and in xanthorhodopsin. The results not only disclose extremely fast energy transfer rates and pathways, they also reveal effects of the binding site on the excited-state properties of the carotenoid. We compared the excited-state dynamics of salinixanthin in xanthorhodopsin and in NaBH₄-treated xanthorhodopsin. The NaBH₄ treatment prevents energy transfer without perturbing the carotenoid binding site, and allows observation of changes in salinixanthin excited-state dynamics related to specific binding. The S₁ lifetimes of salinixanthin in untreated and NaBH₄-treated xanthorhodopsin were identical (3 ps), confirming the absence of the S₁-mediated energy transfer. The kinetics of salinixanthin S₂ decay probed in the near-infrared region demonstrated a change of the S₂ lifetime from 66 fs in untreated xanthorhodopsin to 110 fs in the NaBH₄-treated protein. This corresponds to a salinixanthin-retinal energy transfer time of 165 fs and an efficiency of 40%. In addition, binding of salinixanthin to xanthorhodopsin increases the population of the S^∗ state that decays in 6 ps predominantly to the ground state, but a small fraction (<10%) of the S^∗ state generates a triplet state.     

The Telomeric dG(GT)4G Sequence Can Adopt a Parallel-Stranded Double Helical Conformation

Abstract

Oligonucleotides 3′-d(GTGTGTGTGG)-L-d(GGTGTGTGTG)-3′ (hp-GT) and 3′- d(G^4STG^4STG^4STG^4STGG)-L-d(GGTGTGTGTG)-3′ (hp-SGT), (L=(CH₂CH₂O)₃), were shown by use of several optical techniques to form a novel parallel-stranded (ps) intramolecular double helix with purine-purine and pyrimidine-pyrimidine base pairing. The rotational relaxation time of hp-GT was similar to that of a 10-bp reference duplex, and the fraction of unpaired bases was determined to be ～ 7%, testifying to the formation of an intramolecular double helical hairpin by the sequence under the given experimental conditions. A quasi-two- state mode of ps-double helix formation was validated, yielding a helix-coil transition enthalpy of ?135±5 kJ/mol. The G-G and T·T (or ^4ST·T) base pair configurations and conformational parameters of the double helix were derived with molecular modeling by force field techniques. Repetitive d(GT) sequences are abundant in telomers of different genomes and in the regulatory regions of genes. Thus, the observed conformational potential of the repetitive d(GT) sequence may be of importance in the regulation of cell processes.     

Structural characterization of the L0 cytoplasmic loop of human multidrug resistance protein 6 (MRP6)

ABCC6 is a member of the C subfamily of ATP-binding cassette transporters whose mutations are correlated to Pseudoxanthoma elasticum, an autosomal recessive, progressive disorder characterized by ectopic mineralization and fragmentation of elastic fibers. Structural studies of the entire protein have been hindered by its large size, membrane association, and domain complexity. Studies previously performed have contributed to shed light on the structure and function of the nucleotide binding domains and of the N-terminal region. Here we report the expression in E. coli of the polypeptide E₂₀₅-G₂₇₉ contained in the cytoplasmic L0 loop. For the first time structural studies in solution were performed. Far-UV CD spectra showed that L0 is structured, assuming predominantly α-helix in TFE solution and turns in phosphate buffer. Fluorescence spectra indicated some flexibility of the regions containing aromatic residues. ¹H NMR spectroscopy identified three helical regions separated by more flexible regions.     

Conformational properties of the isolated 1–23 fragment of human hemoglobin α-chain

With the purpose of establishing whether, as a general rule, regions of a protein chain that are helical in the native structure maintain, at least partially, the same helical structure when isolated in solution, we have prepared the 1–23 fragment of human hemoglobin α-chain, and studied its conformational properties in aqueous solution by CD and¹H-NMR. From the analysis of CD and NMR spectral changes with temperature, salt and addition of trifluoroethanol (TFE) it can be concluded that the 1–23 peptide forms a measurable population (18% at 22°C (pH 5.6) TFE/H₂O, 30:70 (v/v)) of an α-helix structure that spans the same residues that are helical in the native protein (namely, 6 to 17). These results, taken together with similar ones obtained previously in the 1–19, 21–42 and 50–61 RNAase fragments, support the idea that no helices other than the native ones are actually formed in solution by protein fragments. This implies that the final helical structure of a protein is present from the very beginning of the folding process, and also that such elements of secondary structure can act as primary nucleation centers.     

Conformational changes of alpha-chymotrypsin in a fibrillation-promoting condition: a molecular dynamics study

Amyloid nanofibril formation appears to be a generic property of polypeptide chains. α-Chymotrypsin (aCT) was recently driven toward amyloid-like aggregation by the addition of trifluoroethanol (TFE) at intermediate concentrations. In this study we employed a molecular dynamics simulation to investigate the early events in TFE-induced conformational changes of aCT that precede amyloid formation, and compared the results of the simulation with previous experiments. TFE molecules were found to rapidly replace the water molecules closely associated with the protein surface. The gyration radius, together with total and hydrophobic solvent-accessible surface areas of aCT, was significantly increased. In accord with the experimental observations, the extended β-conformation of backbone was increased. The secondary structural elements of aCT in water and TFE/water mixture showed a reasonable fit, whereas significant deviations were observed for several loops. These alterations originated largely from main-chain rotations at glycine residues. The catalytic active site and S1 binding pocket of the enzyme were also distorted in the TFE/water mixture. The obtained results are suggested to provide more insights into the conformational properties of the amyloid aggregation-prone protein species. Possible mechanisms of TFE-induced alterations in the conformation and dynamics of the protein structure are also discussed.     

Ultrafast multi-pulse transient absorption spectroscopy of fucoxanthin chlorophyll a protein from Phaeodactylum tricornutum

We have applied femtosecond transient absorption spectroscopy in pump-probe and pump-dump-probe regimes to study energy transfer between fucoxanthin and Chl a in fucoxanthin-Chl a complex from the pennate diatom Phaeodactylum tricornutum. Experiments were carried out at room temperature and 77?K to reveal temperature dependence of energy transfer. At both temperatures, the ultrafast (<100?fs) energy transfer channel from the fucoxanthin S₂ state is active and is complemented by the second pathway via the combined S₁/ICT state. The S₁/ICT-Chl a pathway has two channels, the fast one characterized by sub-picosecond energy transfer, and slow having time constants of 4.5?ps at room temperature and 6.6?ps at 77?K. The overall energy transfer via the S₁/ICT is faster at 77?K, because the fast component gains amplitude upon lowering the temperature. The pump-dump-probe regime, with the dump pulse centered in the spectral region of ICT stimulated emission at 950?nm and applied at 2?ps after excitation, proved that the S₁ and ICT states of fucoxanthin in FCP are individual, yet coupled entities. Analysis of the pump-dump-probe data suggested that the main energy donor in the slow S₁/ICT-Chl a route is the S₁ part of the S₁/ICT potential surface.     

Characterization the Effects of Structure and Energetics of Intermolecular Interactions on the Oligomerization of Peptides in Aqueous 2, 2, 2-Trifluoroethanol via Circular Dichroism and Nuclear Magnetic Resonance Spectroscopy

Intermolecular interactions are of fundamental importance to fully comprehend a wide range of protein behaviors such as oligomerization, folding and recognition. Two peptides, NPY^[18−36] and NPY^[15−29], segmented from human neuropeptide Y (hNPY), were synthesized in this work to study the interaction between species. Information about intermolecular interactions was extracted from their oligomerizing behaviors. The results from CD and NMR showed that the addition of 2, 2, 2-trifluoroethanol (TFE) induces a stable helix in each peptides and an extended helix in NPY^[18−36], formed between residues 30-36. Pulsed field gradient NMR data revealed that NPY^[15−29] forms a larger oligomer at lower temperatures and continuously dissociates into the monomeric form with increasing temperature. NPY^[18−36] was also found to undergo an enhanced interaction with TFE and a more favorable self-association at higher temperatures. We characterized the changes of oligomerized states with respect to temperature to infer the effects of entropy and interaction energy on the association-dissociation equilibrium. As shown by NPY^[15−29], deletion of helical secondary structure or residues from the C-terminal segment may disrupt the solvation by TFE and results in entropy increase as the oligomer dissociates. Unlike that in NPY^[15−29], the extended helix in NPY^[18−36] improves the binding of TFE, and as a result, entropy is gained via the transfer of the TFE cluster from the interface between monomeric peptides into the bulk solvent. This observation suggests that the oligomerized state may be modulated by the entropy and energetics contributed by helical segments in the oligomerization process.     

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {¹H}-¹⁵N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pK_a values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/β proteins both on fast ps/ns as well as slower μs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.     

Utilization of blue-green light by chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus: Ultrafast excitation energy conversion and transfer

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 10⁴–10⁵ bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S₂ state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400–900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret → Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100–270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S₁ state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret → Q_y internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.     

Influence of trifluoroethanol on membrane interfacial anchoring interactions of transmembrane alpha-helical peptides

Interfacial anchoring interactions between aromatic amino acid residues and the lipid-water interface are believed to be important determinants for membrane protein structure and function. Thus, it is possible that molecules that partition into the lipid-water interface can influence membrane protein activity simply by interfering with these anchoring interactions. Here we tested this hypothesis by investigating the effects of 2,2,2-trifluoroethanol (TFE) on the interaction of a Trp-flanked synthetic transmembrane peptide (acetyl-GW₂(LA)₈LW₂A-NH₂) with model membranes of dimyristoylphosphatidylcholine. Two striking observations were made. First, using ²H nuclear magnetic resonance on acyl chain deuterated lipids, we found that addition of 4 or 8 vol % of TFE completely abolishes the ability of the peptide to order and stretch the lipid acyl chains in these relatively thin bilayers. Second, we observed that addition of 8 vol % TFE reduces the tilt angle of the peptide from 5.3° to 2.5°, as measured by ²H NMR on Ala-d₄ labeled peptides. The “straightening” of the peptide was accompanied by an increased exposure of Trp to the aqueous phase, as shown by Trp-fluorescence quenching experiments using acrylamide. The observation of a reduced tilt angle was surprising because we also found that TFE partioning results in a significant thinning of the membrane, which would increase the extent of hydrophobic mismatch. In contrast to the Trp-flanked peptide, no effect of TFE was observed on the interaction of a Lys-flanked analog (acetyl-GK₂(LA)₈LK₂A-NH₂) with the lipid bilayer. These results emphasize the importance of interfacial anchoring interactions for membrane organization and provide new insights into how molecules such as TFE that can act as anesthetics may affect the behavior of membrane proteins that are enriched in aromatic amino acids at the lipid-water interface.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

Structural studies of a synthetic peptide derived from the carboxyl-terminal domain of RNA polymerase II

The conformation of the repeating heptapeptide unit of the carboxyl-terminal domain of RNA Polymerase II, Y¹S²P³T⁴S⁵P⁶S⁷ has been examined using nuclear magnetic resonance spectroscopy and circular dichroism. Nuclear Overhauser effects and CD spectra for the synthetic 56-residue peptide H₂N-(S²P³T⁴S⁵P⁶S⁷Y^l)₈-COOH in water indicate that the peptide is largely unordered. A small population of folded molecules is observed to contain β-turns located at Ser²-Pro³-Thr⁴-Ser⁵ (SPTS) and Ser⁵-Pro⁶-Ser⁷-Tyr¹ (SPSY). CD and NMR results in 90% TFE also indicate an equilibrium population of structures, but the fraction of turns is higher. Similarities of nuclear Overhauser effects in water and in 90% TFE suggest that the structures in TFE are biologically relevant. Based on these observations, the average structure of a single conformer of the heptapeptide repeat in 90% TFE was obtained by a distance geometry-simulated annealing method, using distance restraints extracted from nuclear Overhauser data. NMR spectra of the 56-mer show signals corresponding to only one repeat indicating that each repeat is in an identical environment. Thus it is possible to obtain an average structure of the heptapeptide repeat from NOE data on the 56-mer. Twenty-seven final structures were calculated and the root mean square deviations between the 27 structure and the mean coordinates was 1.52 Å for the backbone and 2.2 Å for all nonhydrogen atoms. The heptapeptide repeat consists of two overlapping β-turns which are potentially stabilized by hydrogen bonds. The hydroxyl side chains of Ser², Ser⁵, Thr⁴, and Ser⁷ all appear to be equally exposed for potential phosphorylation. The tyrosyl side chain of each repeat is folded inwards to the backbone and can potentially hydrogen bond to the carbonyl oxygen of the tyrosine in the preceding repeat. Iteration of the average structure of the heptapeptide repeat results in a model of the carboxyl-terminal domain with a regular but unusual secondary structure consisting of a series of staggered β-turns. © 1995 Wiley-Liss, Inc.     

Segmental conformational disorder and dynamics in the intrinsically disordered protein α-synuclein and its chain length dependence

Conformational ensembles of fully disordered natural polypeptides represent the starting point of protein refolding initiated by transfer to folding conditions. Thus, understanding the transient properties and dimensions of such peptides under folding conditions is a necessary step in the understanding of their subsequent folding behavior. Such ensembles can also undergo alternative folding and form amyloid structures, which are involved in many neurological degenerative diseases. Here, we performed a structural study of this initial state using time-resolved fluorescence resonance energy transfer analysis of a series of eight partially overlapping double-labeled chain segments of the N-terminal and NAC domains of the α-synuclein molecule. The distributions of end-to-end distance and segmental intramolecular diffusion coefficients were simultaneously determined for eight labeled chain segments. We used the coefficient of variation, C_v, as a measure of the conformational heterogeneity (i.e., structural disorder). With the exception of two segments, the C_vs were characteristic of a fully disordered state of the chain. Subtle deviations from this behavior at the segment labeled in the NAC domain and the segment at the N termini reflected subtle conformational bias that might be related to the initiation of transition to amyloid aggregates. The chain length dependence of the mean segmental end-to-end distance followed a power law as predicted by Flory, but the dependence was steeper than previously predicted, probably due to the contribution of the excluded volume effect, which is more dominant for shorter-chain segments. The observed intramolecular diffusion coefficients (< 10 to ∼ 25 ?²/ns) are only an order of magnitude lower than the common diffusion coefficients of low molecular weight probes. This diffusion coefficient increased with chain length, probably due to the cumulative contributions of minor bond rotations along the chain. These results gave us a reference both for characteristics of a natural unfolded polypeptide at the moment of initiation of folding and for detection of possible initiation sites of the amyloid transition.