Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study

The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.     

UV resonance Raman determination of protein acid denaturation: selective unfolding of helical segments of horse myoglobin

We have used UV resonance Raman spectroscopy to study the acid denaturation of horse heart aquometmyoglobin (Mb) between pH 7.5 and 1.5. Raman spectra excited at 206.5 nm are dominated by amide vibrations, which are analyzed by using a new methodology to quantitatively determine the Mb secondary structure. In contrast, the 229-nm Raman spectra are dominated by the Tyr and Trp Raman bands, which are analyzed to examine changes in Tyr and Trp environments, such as exposure to water, hydrogen bonding, and, for Trp, any alterations of the dihedral angle between the Trp ring and its linkage to the protein backbone. We uniquely determined which Mb alpha-helices melt by combining the amide, Tyr, and Trp Raman spectral information with heme absorption spectral information. We calculate that the Mb alpha-helical composition decreases from approximately 80% at neutral pH to approximately 19% below pH 3.5. The Trp Raman cross sections dramatically decrease at low pH to values which indicate that they are fully exposed to water; this result indicates that the A helix melts. The Tyr Raman bands are pH independent, which indicates that the G and H helices around the Tyr residues do not melt. The dramatic heme absorption acid denaturation changes indicate major alterations of the heme pocket and changes in heme binding. These results indicate that the A, B, C, D, E, and F helices melt in a concerted fashion, while the antiparallel G and H helices only partially melt.     

Amide modes of the alpha-helix: Raman spectroscopy of filamentous virus fd containing peptide 13C and 2H labels in coat protein subunits

The filamentous virus fd consists of a single-stranded DNA genome sheathed by 2700 copies of a 50-residue alpha-helical subunit (protein pVIII) and serves as a model assembly of alpha-helices. To advance vibrational assignments for the alpha-helix, we have investigated Raman spectra of fd virions containing 13C and 2H (deuterium) labels at various main-chain sites of the pVIII subunits. 13C was introduced at specific peptide carbonyls, while deuterium was introduced at selected alpha-carbon (Calpha) and amide nitrogen positions. Interpretation of the Raman spectra reveals a previously unrecognized alpha-helix band in the spectral interval 730-745 cm-1, tentatively assigned to a carbonyl in-plane bending mode (amide IV). Experimental evidence has also been obtained for a distinctive alpha-helix marker near 1345 cm-1, assigned to a coupled Calpha-H bending and Calpha-C stretching mode. The fd virions containing 13C-labeled carbonyls exhibit unexpectedly complex amide I profiles, consisting of multiple band components. Amide I splitting resulting from 13C substitution of carbonyls is attributed to decoupling of transition-dipole interactions normally occurring in the extended pVIII helix. The present study identifies novel conformation-dependent Raman bands in a native alpha-helix assembly, confirms amide I and amide III assignments proposed previously for filamentous viruses, and facilitates new Raman assignments for the packaged ssDNA. The alpha-helix markers identified here should also be useful in conformation analyses of other proteins by Raman spectroscopy.     

Structure and thermal stability of photosystem II reaction centers studied by infrared spectroscopy

The secondary structure of photosystem II reaction centers isolated from pea has been deduced from quantitative analysis of the component bands of the infrared amide I spectral region, determined by FTIR spectroscopy. The analysis shows the isolated complex to consist of 40% alpha-helix, 10% beta-sheet, 14% beta-strands (or extended chains), 17% turns, 15% loops, and 3% nonordered segments. These structural protein elements were determined for samples in H2O, in D2O, and in dried films. The isolated reaction center, composed of proteins D1,D2,cytochrome b559, and PsbI, has been predicted to contain a total of 13 transmembrane alpha-helices, which conveys a percentage of this type of structure congruent with the structural determination deduced from FTIR spectra. The process of thermal destabilization of the reaction centers has also been studied by FTIR spectroscopy, showing a clear main conformational transition at 42 degrees C, which indicates a high thermal sensitivity of the secondary structure of this protein complex. Such thermal instability may correlate with the well-described high sensitivity of photosystem II to damage and may relate to the process of rapid protein degradation that photosystem II suffers during photoinhibition of plants.     

Determination of secondary structure of normal fibrin from human peripheral blood

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% alpha-helix (amide I and III components at 1653 cm-1, and 1312 and 1284 cm-1, respectively), 40% beta-sheets (amide I and III components at 1625 and 1231 cm-1, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm-1, and 1249 cm-1, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is < 10% for percentage values of amide I and III components > 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin beta-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (alpha-helix, beta-sheets, and turns) are accurate.     

Strain-dependent differences in beta-sheet conformations of abnormal prion protein

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm-1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 cm-1 and stronger band intensity at both 1616 and 1694-1695 cm-1. Because absorbances from 1616 to 1636 cm-1 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.     

Effect of ethanol on the protein secondary structure of the human gastric mucosa, in vitro

The effect of ethanol on the secondary conformational structure of proteins of the human gastric mucosa was investigated by attenuated total reflection/Fourier transform infrared (ATR/FT-IR) spectroscopy. The IR peak intensity and position of each structural component of gastric mucosa was found to change significantly with the ethanol concentration and length of exposure. The peak intensity due to the beta-sheet and/or beta-turn conformational structure in amide I and II bands of gastric mucosa clearly increased after treatment with ethanol. Moreover, the peak at 1635 cm-1 shifted to 1630 cm-1 after treatment with 40% ethanol for 3 h, or 80% ethanol for 1 h, and a distinct shoulder also appeared at 1643 cm-1. This shift occurred more rapidly and was more pronounced after exposure of mucosa to 80% ethanol, compared with the effect of 40% ethanol, but the alpha-helical structure at the amide I and II bands was not influenced by either concentration of ethanol. Ethanol treatment might also transform the secondary structure of amide III in gastric mucosa from an alpha-helix to a mainly random coil with extensive unfolding. The absorption between 1180 and 980 cm-1, which is assigned to glycoprotein structure, was also reduced after treatment with ethanol. This strongly indicates that ethanol influences the conformation of the lipids and proteins of human gastric mucosa, leading to their deformation.     

Thermally denatured ribonuclease A retains secondary structure as shown by FTIR

Fourier transform-infrared (FTIR) spectroscopy has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A) at pH* 2.0 (uncorrected pH measured in D2O). The amide I spectral region of the native and thermally denatured protein was compared. A substantial decrease in the amount of beta-sheet and alpha-helix and a corresponding increase in the amount of turn and unordered structure was observed on thermal denaturation. The results indicate that thermally denatured RNase A contains significant amounts of secondary structure (11% helix and 17% beta-sheet), consistent with previous results reported for circular dichroism, and with a relatively compact structure, as revealed by dynamic light scattering. These results are in contrast to those of amide protection experiments reported recently [Robertson, A.D., & Baldwin, R.L. (1991) Biochemistry 30, 9907-9914] which indicated no stable hydrogen-bonded structure under these experimental conditions. Possible explanations for this apparent discrepancy are given.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

Structural analysis of a hmg-coA-reductase pseudogene: insights into evolutionary processes affecting the hmgr gene family in allotetraploid cotton (Gossypium hirsutum L.)

Fourier transform infrared spectroscopy has been used to investigate the conformational changes of glycinin. a major storage protein of soybean seeds, upon film-forming. The results show that the secondary structure of glycinin is mainly composed of a beta-sheet (48%) and unordered (49%) structures. The amide I band of glycinin in film-forming conditions, i.e. in alkaline media and in the presence of plasticizing agent, reveals the conversion of 18% of the secondary structure of the protein from the beta-sheet (6%) and random coil (12%) to the alpha-helical conformation due to the helicogenic effect of the ethylene glycol used as the plasticizing agent. Conformational changes also occur upon the film-forming process leading to the formation of intermolecular hydrogen-bonded beta-sheet structures. Results obtained from other plant families indicate that, whatever the origin and conformation of protein, formation of films leads to the appearance of intermolecular hydrogen-bonded beta-sheet structures, suggesting that this type of structure might be essential for the network formation in films. Thus, it is hypothesized that, in the film state, intermolecular hydrogen bonding between segments of beta-sheet may act as junction zones in the film network. This study reveals for the first time that there is a close relationship between the conformation of proteins and the mechanical properties of films.     

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: acid-induced denaturation

Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or "A" state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.     

A new approach to secondary structure evaluation: secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover alpha-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing alpha-helical structures. For segments with dominating beta-sheet conformation, however, the application of this method is limited due to the stability and clustering of beta-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed beta-sheet CD signals. Nevertheless, the results of this method especially for alpha-helical segments are very impressive. All alpha-helical and 71% of the beta-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of alpha-helical secondary structure elements and help localizing them on the sequence string.     

Laser Raman investigation of the conformation of human immunoglobulin G

Laser Raman spectra of human immunoglobulin G in neutral solution, as well as in the lyophilized and alkaline-denatured states are presented. In the spectrum of the native protein, the amide III band appears at 1240 cm-1 and is assigned to the presence of beta-sheet structure. From its intensity, using a procedure described in this paper, we evaluate the beta-structure content to 37 +/- 4%. This result is supported by the strong amide I' band at 1667 cm-1 and by the presence in the spectra of two bands at 991 and 1078 cm-1, respectively assigned to the C-C and C-N skeletal stretching modes. The differences between the spectrum of the lyophilized powder and that of the solution show that the lyophilization process induces conformational changes that perturb the local environment of some of the tryptophan residues and alter the secondary structure of immunoglobulin G. The beta-structure appears to be more uniform and more abundant in solution. When the protein is denatured at pH 11, the amide III and amide I'bands, which become weaker and broader, shift in frequency from 1240 to 1248 cm-1 and from 1667 to 1656 cm-1 respectively. These changes indicate a decrease in the amount of beta-structure and a transition toward a much more disordered conformation. During the denaturation, the intensities of many bands of the aromatic chromophores change, notably the tryptophan peaks at 879, 1359 and 1573 cm-1.     

Secondary structure analysis of individual transmembrane segments of the nicotinic acetylcholine receptor by circular dichroism and Fourier transform infrared spectroscopy

Circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure of peptides containing one or more transmembrane segments (M1-M4) of the Torpedo californica nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3 and M1-M2-M3 transmembrane segments of the AChR beta-subunit and the M4 segment of the alpha- and gamma-subunits were isolated from proteolytic digests of receptor subunits, purified, and reconstituted into lipid vesicles. For each peptide, an amide I vibrational frequency centered between 1650 and 1656 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical. In addition, the CD spectrum of a tryptic peptide of the alpha-subunit containing the M1 segment is also consistent with a largely alpha-helical structure. However, secondary structure analysis of the alpha-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted alpha-helix, likely the consequence of several proline residues, or may not be entirely alpha-helical. Overall, these findings are consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spanning alpha-helices.     

Three-dimensional structure of leucocin A in trifluoroethanol and dodecylphosphocholine micelles: spatial location of residues critical for biological activity in type IIa bacteriocins from lactic acid bacteria

The first three-dimensional structure of a type IIa bacteriocin from lactic acid bacteria is reported. Complete 1H resonance assignments of leucocin A, a 37 amino acid antimicrobial peptide isolated from the lactic acid bacterium Leuconostoc gelidum UAL187, were determined in 90% trifluoroethanol (TFE)-water and in aqueous dodecylphosphocholine (DPC) micelles (1:40 ratio of leucocin A:DPC) using two-dimensional NMR techniques (e.g., DQF-COSY, TOCSY, NOESY). Circular dichroism spectra, NMR chemical shift indices, amide hydrogen exchange rates, and long-range nuclear Overhauser effects indicate that leucocin A adopts a reasonably well defined structure in both TFE and DPC micelle environments but exists as a random coil in water or aqueous DMSO. Distance geometry and simulated annealing calculations were employed to generate structures for leucocin A in both lipophilic media. While some differences were noted between the structures calculated for the two different solvent systems, in both, the region encompassing residues 17-31 assumes an essentially identical amphiphilic alpha-helix conformation. A three-strand antiparallel beta-sheet domain (residues 2-16), anchored by the disulfide bridge, is also observed in both media. In TFE, these two regions have a more defined relationship relative to each other, while, in DPC micelles, the C-terminus is folded back onto the alpha-helix. The implications of these structural features with regard to the antimicrobial mechanism of action and target recognition are discussed.     

Solution structure of the origin DNA-binding domain of SV40 T-antigen

The structure of the domain from simian virus 40 (SV40) large T-antigen that binds to the SV40 origin of DNA replication (T-ag-OBD131-260) has been determined by nuclear magnetic resonance spectroscopy. The overall fold, consisting of a central five-stranded antiparallel beta-sheet flanked by two alpha-helices on one side and one alpha-helix and one 3(10)-helix on the other, is a new one. Previous mutational analyses have identified two elements, termed A (approximately 152-155) and B2 (203-207), as essential for origin-specific recognition. These elements form two closely juxtaposed loops that define a continuous surface on the protein. The addition of a duplex oligonucleotide containing the origin recognition pentanucleotide GAGGC induces chemical shift changes and slows amide proton exchange in resonances from this region, indicating that this surface directly contacts the DNA.     

Effect of guanidine hydrochloride on the holo- and apo-enzymes of pig heart lipoamide dehydrogenase

1. The effect of guanidine hydrochloride (GuHCl) on pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3.] was investigated by means of enzymatic activity and optical measurements (CD, absorption, and fluorescence spectra). The activity of the enzyme decreased on increasing the concentration of GuHCl and the enzyme was completely inactivated in 2.0 M GuHCl. 2. The contents of alpha-helix, beta, and unordered forms in lipoamide dehydrogenase were estimated to be 34, 14, and 52%, respectively. On increasing the concentration of GuHCl, the content of alpha-helix in lipoamide dehydrogenase decreased, whereas the content of the beta form hardly changed. 3. The native lipoamide dehydrogenase showed absorption, CD, and fluorescence spectra characteristic of bound FAD in the visible region, suggesting hydrophobic interaction between the protein moiety and FAD chromophore. The absorption, CD, and fluorescence spectra of the enzyme in 2.0 M GuHCl were similar to those of free FAD in the buffer, suggesting the release of FAD from the protein moiety. 4. The protein fluorescence spectrum of lipoamide dehydrogenase had a maximum at 350 nm blue-shifted by 8 nm from that of tryptophan in aqueous solution. The maximum of the enzyme in 2.0 M GuHCl was red-shifted to 357 nm. This suggests exposure of tryptophan residues to a polar environment. The maximum, 352nm, of the apoenzyme shifted to 350 nm on addition of FAD. These results show that the conformation in the microenvironment of some tryptophan residues in lipoamide dehydrogenase is affected by the dissociation-association of FAD. 5. The contents of alpha-helix, beta, and unordered forms in the apoenzyme were estimated to be 35, 8, and 57%, respectively. These values are similar to those of the native holoenzyme. The alpha-helical structure in the apoenzyme molecule was more sensitive to GuHCl than that in the holoenzyme. FAD and two hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4 benzolamido-4'-aminostilbene-2,2'-disulfonate (MBAS), which can bind to the apoenzyme, stabilized the alpha-helical structure in the apoenzyme molecule.     

Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L

The 1H NMR assignments have been made for the immunoglobulin (Ig) light chain-binding B1 domain of protein L from Peptostreptococcus magnus. The secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone coupling constants, and slowly exchanging amide protons. The B1 domain was found to be folded into a globular unit of 61 amino acid residues, preceded by a 15 amino acid long disordered N-terminus. The folded portion of the molecule contains a four-stranded beta-sheet spanned by a central alpha-helix. The fold is similar to the IgG-binding domains of streptococcal protein G, despite the fact that the binding sites on immunoglobulins for the two proteins are different; protein G binds IgG through the constant (Fc) part of the heavy chain, whereas protein L has affinity for the variable domain of Ig light chains.     

Secondary structure of P-glycoprotein investigated by circular dichroism and amino acid sequence analysis

P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp.