pH dependence of amide chemical shifts in natively disordered polypeptides detects medium-range interactions with ionizable residues

A growing number of natively disordered proteins undergo a folding/binding process that is essential for their biological function. An interesting question is whether these proteins have incompletely solvated regions that drive the folding/binding process. Although the presence of predominantly hydrophobic buried regions can be easily ascertained by high-sensitivity differential scanning calorimetry analysis, the identification of those residues implicated in the burial requires NMR analysis. We have selected a partially solvated natively disordered fragment of Escherichia coli, thioredoxin, C37 (38-108), for full NMR spectral assignment. The secondary chemical shifts, temperature coefficients, and relaxation rates (R(1) and R(2)) of this fragment indicate the presence of a flexible backbone without a stable hydrogen bond network near neutral pH. (1)H-(15)N heteronuclear single quantum coherence analysis of the pH dependence of amide chemical shifts in fragment C37 within pH 2.0 and 7.0 suggests the presence of interactions between nonionizable residues and the carboxylate groups of four Asp and four Glu residues. The pH midpoints (pH(m)) of the amides in the ionizable residues (Asp or Glu) and, consequently, the shifts in the pH(m) (DeltapH(m)) of these residues with respect to model tetrapeptides, are sequence-dependent; and the nonionizable residues that show pH dependence cluster around the ionizable ones. The same pH dependence has been observed in two fragments: M37 (38-73) and C73 (74-108), ruling out the participation of long-range interactions. Our studies indicate the presence of a 15-residue pH-dependent segment with the highest density of ionizable sites in the disordered ensembles of fragments C37 and M37. The observed correlations between ionizable and nonionizable residues in this segment suggest the organization of the backbone and side chains through local and medium-range interactions up to nine residues apart, in contrast to only a few interactions in fragment C73. These results agree qualitatively with the predominantly hydrophobic buried surface detected only in fragments C37 and M37 by highly sensitive differential scanning calorimetry analysis. This work offers a sensitive and rapid new tool to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the growing family of natively disordered small proteins with full NMR assignments.     

Noncovalent complex between domain AB and domains CD*EF of parvalbumin

The interaction between domain AB and domains CD^*EF of pike parvalbumin III has been studied by intrinsic fluorescence spectroscopy. In the presence of Ca²⁺ ions, parvalbumin fragment 38–108 containing two calcium binding sites interacts with the short peptide 1–37 with association constant 10^5.3±0.5M⁻¹. Removal of Ca²⁺ ions results in the disappearance of the interaction. The affinity of the complex of the two fragments for calcium is 50-times higher than the affinity of the isolated fragment 38–108, but slightly lower than that of the intact protein.     

Conformations of isolated fragments of pancreatic polypeptide

In spite of its short polypeptide chain, the pancreatic polypeptide molecule consists of a polyproline II type helix and alpha-helix. To understand the stability and formation of the alpha-helical region, we prepared some peptide fragments including the helical segment of chicken pancreatic polypeptide and studied their conformations by circular dichroism (CD). PP7-36 (a peptide fragment corresponding to residues 7-36 of chicken pancreatic polypeptide) showed a CD spectrum characteristic of the helix at pH 4.6 and at peptide concentrations as low as 1 microM. PP11-36 was able to form a helical conformation only at high peptide concentrations and not at concentrations lower than 10 microM. However, acetyl PP11-36 (in which the alpha-amino group is acetylated so that no positive charge exists at the N terminus) was able to form the helical conformation at pH 4.6 and at the peptide concentrations where PP11-36 could not. Succinyl PP12-36 (in which the alpha-amino group is succinylated to introduce a negative charge) was also able to form the helical conformation. The CD spectra of PP12-36 and PP13-36 were not characteristic of the helical conformation at all the pH values and peptide concentrations studied. Acetyl PP13-36, which has no charge at the N terminus, did not form the helix. On the other hand, succinyl PP13-36, which has a negative charge at the N-terminal end, did form the helix at pH 4.6. These findings indicate that the presence of the negative charge of carboxylate at the N-terminal region of a peptide fragment is important for helix formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

DSC studies of a family of natively disordered fragments from Escherichia coli thioredoxin: surface burial in intrinsic coils

The accumulating data from proteome analysis indicates that numerous proteins have segments and/or domains, involved in regulatory functions of the eukaryotic cell, which are entirely unstructured under physiological conditions, challenging the structure-function paradigm. Although many such natively unfolded proteins have been structurally analyzed by NMR spectroscopy, little is known about solvent inaccessible surfaces in premolten globules and intrinsic coils. Recent DSC studies of two protein fragments have shown a promising way to estimate the predominantly hydrophobic buried surfaces [Georgescu, R. E., García-Mira, M. M., Tasayco, M. L., and Sánchez-Ruiz, J. M. (2001) Eur. J. Biochem. 268, 1-10]. Here we report a systematic heat capacity analysis of a family of natively disordered complementary fragments of oxidized Escherichia coli thioredoxin (1-31/32-108, 1-37/38-108, 1-50/51-108, and 38-73) which provides insights into the local and nonlocal interactions contributing to the burial of predominantly hydrophobic surface in intrinsic coils.     

Structure and enzymatic functions of thioredoxin refolded by complementation of two tryptic peptide fragments.

The physicochemical and catalytic properties of thioredoxin-T' are described. This complemented protein structure consists of a 1:1 complex between the inactive fragments thioredoxin-T-(1--73) and thioredoxin T-(74--108). These are generated by selective trypsin cleavage at Arg-73 in lysine-modified and denatured Escherichia coli thioredoxin. Thioredoxin-T' was a slowly formed but stable complex with an apparent KD below 10(-8) M. The tryptophan fluorescence spectrum and the CD spectrum were very similar to those of native thioredoxin; some conformational differences were detected by gel chromatography and radioimmunoassay. Thioredoxin-T'-S2 was a substrate for NADPH and thioredoxin reductase and had 1--2% of the activity of native thioredoxin. This low relative activity was the result of a major increase in the Km value. Thioredoxin-(SH)2 was a hydrogen donor for E. coli ribonucleotide reductase with about 3% relative activity. These results for thioredoxin-T' are correlated with the known three-dimensional structure of thioredoxin. The microenvironment around Arg-73 that is close to the active disulfide appears to be of critical importance for the interactions of thioredoxin with thioredoxin reductase and ribonucleotide reductase.     

Domain characteristics of the carboxyl-terminal fragment 206–316 of thermolysin: pH and ionic strength dependence of conformation

The pH and ionic strength dependence of conformation of the COOH-terminal fragment 206–316 (fragment FII) of thermolysin was monitored by far-uv CD and difference absorption measurements. This fragment was shown previously to possess the properties of a protein domain, i.e., able to refold into a stable nativelike structure [Fontana, A., Vita, C. & Chaiken, I. M. (1983) Biopolymers 22 , 69–78]. Analysis of the CD spectra in the pH range of 1–12 indicated that near pH 1, the conformation of fragment FII appears to be in an intermediate state (H) between the fully unfolded one (U) [the guanidine hydrochloride (Gdn · HCl)-induced unfolded state] and the nativelike state (N—that attained at neutral pH). Quantitative analysis of secondary structure from CD spectra revealed that state H at 4°C is characterized by some 30% α-helical structure, compared to 47% for state N. The heat- and Gdn · HCl-mediated unfolding transitions of state H were fully reversible and characterized by little cooperativity, which is taken as an indication that state H corresponds to several species possessing different, and low, conformational stabilities. The midpoint transition from state H to N occurs near pH 2.5, implying that the acid transition results from the titration of carboxyl groups of the fragment with anomalously low pK, as would be expected for groups involved in specific salt bridges. Fragment FII at pH 1 (state H) may be induced to exhibit nearly the same degree of helicity of state N simply by increasing the ionic strength of the solution, thus reducing the repulsive interactions between positive charges within the highly charged fragment at pH 1. The results obtained emphasize the role of electrostatic interactions in the folding and stability of fragment FII and suggest a mechanism of folding of the fragment from U to N involving an intermediate state characterized by an assembly of fluctuating α-helices.     

Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments

Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively).     

Characterization of two Cu-containing protein fragments obtained by limited proteolysis of Hyphomicrobiumdenitrificans A3151 nitrite reductase

The unusual Hyphomicrobium denitrificans nitrite reductase containing two type 1 Cu sites and one type 2 Cu site (MW, 50 kDa) has been proteolyzed to two protein fragments (14 and 35 kDa) with subtilisin. The visible absorption, CD, and EPR spectra of these proteins imply that the blue 14-kDa protein fragment has one type 1 Cu site, which is axially elongated trigonal bipyramidal, and the green 35-kDa protein fragment has one type 1 Cu site having a flattened tetrahedral geometry with one type 2 Cu site. The 35-kDa fragment shows the nitrite reduction activity a little higher than to that of native HdNIR. The redox potentials of the 14- and 35-kDa fragments are +345 and +353mV vs. NHE at pH 7.0, respectively. Moreover, the intermolecular electron transfer rate constant of the 35-kDa fragment from an electron donor, cognate cytochrome c(550), is nearly the same as that of the native enzyme.     

Purification and characterization of peptic fragments derived from the carboxyl-terminal half of human albumin

Utilizing a combination of conventional and affinity-chromatographic procedures, we have purified four fragments of human albumin that were generated by controlled limited proteolysis with pepsin [0.3 mM albumin; 37°C; 10 min; pH 3.51; 4.2 mM octanoate; pepsin/albumin, 1:1000 (w/w)]. These fragments have a molecular weight range of 9200-17,000 Da. Amino acid compositions, N- and C-terminal sequences, molecular weights, and other internal markers were used to determine the location of these fragments within the parent molecule. All of the fragments were shown to be derived from the C-terminal half of human albumin. The presence of multiple pepsin-sensitive bonds near the C terminus of each fragment complicated the assignment of specific residue numbers to each fragment. Two pairs of similar peptides were identified: (A) those corresponding to a single-loop structure (residues 309–380 and 309–387) and (B) those containing multiple loops and intraloop cleavages [residues 309–(491–495) with 408–423 deleted]. Purification of these fragments without disulfide bond reduction confirms portions of the loop structure of human albumin and demonstrates increased susceptibility of two specific regions of the C-terminal half of the molecule to peptic digestion.     

Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.     

Conformational study of fragments of envelope proteins (gp120: 254-274 and gp41: 519-541) of HIV-1 by NMR and MD simulations.

The envelope proteins, gp 120 and gp41 of HIV-1, play a crucial role in receptor (CD4+ lymphocytes) binding and membrane fusion. The fragment 254-274 of gp120 is conserved in all strains of HIV and, as a part of the full gp120 protein, behaves as 'immunosilent', but as an individual fragment it is 'immunoreactive'. When this fragment binds to its receptor, it activates the fusion domain of gp41 allowing viral entry into the host CD4+ cells. The conformation of fragment 254-274 of the gp120 domain and fragment 519-541 of the gp41 domain was studied by NMR and MD simulations. The studies were carried out in three varied media--water, DMSO-d6 and hexafluoroacetone (HFA). The fusogenic nature of the gp41 domain peptide was investigated by 31P NMR experiments with model bilayers prepared from dimyristoyl-L-alpha-phosphatidylcholine (DMPC). The solvent was seen to exert a major effect on the structure of the two peptides. Fragment (254-274) of gp120 in DMSO-d6 had a type I beta-turn around the tetrad Val9-Ser10-Thr11-Gln12 while in HFA a helical structure spanning the region Ile5 to Gln12 was seen with the remaining part of the peptide in a random coil structure. It is possible that the beta-turn may constitute an initiation site for the formation of the helix. In water at pH 4.5, the peptide adopted a beta-sheet. The NMR results for fragment 519-541 of gp41 are conclusive of a beta-sheet structure in DMSO-d6, a conformation which may help in insertion into the membrane, a notion also put forward by others. The 31P NMR studies of DMPC vesicles with this fragment show its fusogenic nature, promoting fusion of unilamellar vesicles to larger agglomerates like multilamellar ones.     

Heat capacity analysis of oxidized Escherichia coli thioredoxin fragments (1--73, 74--108) and their noncovalent complex. Evidence for the burial of apolar surface in protein unfolded states.

We have calculated the absolute heat capacities of fragments 1--73 (N fragment) and 74--108 (C fragment) from thioredoxin, their complex and the uncleaved protein, from the concentration dependence of the apparent heat capacities of the solutions determined by differential scanning calorimetry. We find that, while the absolute heat capacities of uncleaved, unfolded thioredoxin and the C fragment are in good agreement with the theoretical values expected for fully solvated chains (calculated as the sum of the contributions of the constituent amino acids), the absolute heat capacities of the N fragment and the unfolded complex are about 2 kJ x K(-1) x mol(-1) lower than the fully solvated-chain values. We attribute this discrepancy to burial of the apolar surface in the N fragment (as burial of the polar area is expected to lead to an increase in heat capacity). Illustrative calculations suggest that burial of about 1000--1600 A(2) of apolar surface takes place in the N fragment (probably accompanied by the burial of a smaller amount of polar surface). In general, this work is supportive of heat capacity measurements on protein fragments being useful as probes of surface burial in studies to characterize protein unfolded states and the high regions of protein folding landscapes.     

Nickel(II) binding to Cap43 protein fragments

Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Synthesis and conformational analysis of salivary proline‐rich peptide P‐B

The 57‐amino acid human salivary polypeptide P‐B has been synthesized by the solid‐phase method using 9‐fluorenylmethoxycarbonyl (Fmoc) strategy. The circular dichroism (CD) spectroscopy, Fourier‐transform infrared spectroscopy (FTIR) and molecular modeling methods have been used for conformational studies of P‐B. Examination of the CD spectra of P‐B showed the content of the secondary structure to be independent of temperature over the range 0–60 °C at pH = 7 as well as over the pH range of 2–12 at 37 °C. P‐B adopts predominantly unordered structure with locally appearing β‐turns. The cumulative results obtained using the CD and FTIR spectroscopic techniques indicate the percentage of the polyproline type‐II (PPII) helix being as low as about 10%. Similarly, the molecular dynamics (MD) simulations reveal only a short PPII helix in the C‐terminal fragment of the peptide (Pro⁵¹–Pro⁵⁴), which constitutes 7%. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of parathyroid hormone fragments produced by cathepsin D

Cleavage of parathyroid hormone by cathepsin D was studied. Four primary products were detected and separated by high performance liquid chromatography. Two of the fragments are fluorescent and therefore contain residue 23 (tryptophan). These fragments are NH2-terminal in origin. The other two cross-react with antisera directed against COOH-terminal portions of the hormone; they are the complementary COOH-terminal fragments. Microsequencing and amino acid analysis showed that the two COOH-terminal fragments are 35-84 and 38-84 bovine parathyroid hormone. By CNBr cleavage and amino acid analysis, the two NH2-terminal fragments were shown to be the complementary 1-37 and 1-34 fragments. The 1-37 fragment is transitory and is rapidly hydrolyzed to 1-34, so that only relatively small amounts are detected at any one time. However, 34-84 was not converted to 38-84, although cleavage at other sites in the COOH-terminal fragments was observed with more exhaustive digestion. The 1-34 fragment appears to be the final product of the action of cathepsin D on parathyroid hormone. Both enzymatically produced NH2-terminal fragments were fully active in the renal membrane adenylyl cyclase assay system.     

Specific encapsidation of fragments of TMV RNA.

The in vitro reconstitution of tobacco mosaic virus (TMV) is initiated by the binding of a disk of TMV protein to the 'disk recognition site', a region of the RNA chain at or near the 5'-terminus for which the disk has special affinity. In order to gain insight into the recognition process, we have studied the ability of disks to encapsidate short RNA fragments produced by partial pancreatic or T1 RNase digestion of TMV RNA. The disk is capable of dicriminating among such fragments, encapsidating only a few of the many present in the digest. The products of encapsidation are short nucleoprotein rods of the same diameter as TMV and of length proportional to that of the encapsidated RNA fragment. The particles differ from TMV, however, in one significant aspect (apart from their length): they possess rings of RNA-free protein at one or both extremities of the rod. In the case of T1 RNase digestion the principal encapsidated fragments were fragments T1 (105 nucleotides) and a family of smaller fragments containing elements of the same sequence. Partial digestion with pancreatic RNase generated only one major fragment (fragment P1; 150 nucleotides) with affinity for the disk. Fragment T1 has been sequenced and shown to represent a portion of the coat protein cistron. Fragment P1 has been partially sequenced but its function is not yet known. Several lines of evidence indicate that fragment T1 is not the disk recognition site. The portion of the TMV RNA chain from which fragment P1 is derived, on the other hand, is encapsidated early in the reconstitution process; thus fragment P1 may contain the disk recognition site. Fragment T1 and fragment P1 both have purine-rich and cytosine-poor sequences near their termini. In addition, fragment T1, and possibly fragment P1, possess a periodicity of order three in purine residues. It seems likely that one or both of the aforesaid properties are largely responsible for the affinity of these fragments for the disk.     

Human pituitary growth hormone: immunochemical investigations of biologically active fragments

Guinea pig antisera to human growth hormone were tested for their ability to recognize the two biologically active fragments of the hormone produced by human plasmin digestion and a synthetic active fragment. A precipitin line was obtained with native human growth hormone, plasmin-treated human growth hormone, and its NH₂-terminal fragment (residues 1–134). In the microcomplement-fixation and radioimmunoassay experiments, the NH₂-terminal plasmin fragment (residues 1–134) showed a greater immunoreactivity than the COOH-terminal plasmin fragment (residues 141–191). This, in turn, was more active than the synthetic fragment (residues 95–136).     

Synthesis of fragments by classical solution methods for use in cytochrome c semisynthesis

We report details of the chemical synthesis of two fragments reproducing the C-terminal sequences 71-108 and 70-108 of Saccharomices cerevisiae cytochrome c. Preparation of the fragments employed classical solution methods and a fragment-condensation strategy; they have been used, together with a third fragment (sequence 67-108) [L. Moroder, B. Filippi, G. Borin & F. Marchiori (1975) Biopolymers 14 , 2061–2074], in the semisynthesis of chimeric cytochromes [C. J. A. Wallace, G. Corradin, F. Marchiori & G. Borin (1986) Biopolymers 25 , 2121–2132].     

Electro-optical analysis of 'curved' DNA fragments

The structure of some 'short' DNA fragments with 106-108 base-pairs and of a 'long' kinetoplast DNA fragment with 419 base-pairs has been analyzed by electro-optical procedures. According to their electrophoretic mobilities and circularization probabilities, it was concluded that two of our short fragments with clusters of four and five and six adenosines phased at the period of the double helix are inherently curved with an approximate curvature around 200 degrees. The dichroism decay curves of our short fragments exhibited two processes. A fast one with time constants of approx. 100 ns is attributed to bending; the bending amplitudes observed for the fragments with dA4 and dA5/6 clusters are slightly higher (23 and 29%, respectively) than those observed for control fragments (17-20%). The second process reflects the overall rotational diffusion of the whole fragments and shows some variation with the DNA sequence, but on average the rotation of fragments with dA4 and dA5/6 clusters corresponds to that observed for standard DNA. Since the rotational diffusion coefficients are very strongly dependent on the effective hydrodynamic lengths, we must conclude that the effective lengths of our fragments, including the 'curved' ones, are very similar under the conditions of our experiments. The rotation time constant for the long kinetoplast DNA is also rather close to those observed for the usual DNA fragments of corresponding length. One way to resolve the conflict of our results with conclusions obtained from other investigations would invoke the assumption that the curved fragments are not 'elastic'. According to this hypothesis, electric field pulses would stretch the curved fragments to an almost straight form and the stretched DNA would return to its equilibrium state with a time constant longer than the rotation time constant.