The crystal and molecular structures of diferric porcine and rabbit serum transferrins at resolutions of 2.15 and 2.60 A, respectively.

The serum transferrins are monomeric proteins with a molecular mass of around 80 kDa and are responsible for the transport of iron in vertebrates. The three-dimensional structures of diferric porcine and rabbit serum transferrin have been refined against X-ray diffraction data extending to 2.15 and 2.60 A, respectively. Data for both proteins were collected using synchrotron radiation at temperatures of 277 K. The porcine protein crystallizes in the space group C2, with unit-cell parameters a = 223.8, b = 44.9, c = 78.9 A, beta = 105.4 degrees with one molecule in the asymmetric unit. The structure was solved by molecular-replacement methods using rabbit serum transferrin as the search model. The structure was refined using REFMAC, with a final residual of 13.8% (R(free) = 18.2% for a 5% data sample) for all data to 2.15 A. The final model comprises 5254 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, one N-acetyl glucosamine moiety and 494 water molecules. The rabbit protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = 127.2, c = 144.9 A and one molecule per asymmetric unit. The structure was solved using the method of multiple isomorphous replacement and refined using REFMAC to give a final residual of 18.6% (R(free) = 22.2% for a 5% data sample) for all data to 2.60 A. The final model comprises 5216 protein atoms, two Fe(3+) cations and two CO(3)(2-) anions, a Cl(-) anion and 206 solvent molecules; there is no clear indication of the carbohydrate moiety attached to Asn490 (rabbit serum numbering). Both molecules adopt a bilobal structure typical for members of the transferrin family. Each of the structurally homologous lobes contains two dissimilar domains with a single iron-binding site buried within the interdomain cleft. The porcine serum protein lacks an interdomain disulfide bridge close to the connecting peptide between the lobes, but this seems to have little effect on the overall orientation of the lobes. The N-lobes of both proteins possess lysine residues, one from each of the two domains, that lie in close proximity to one another to form the so-called dilysine trigger. The more acid-labile release of iron from serum transferrins than from lactoferrins is discussed.

Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I.

We have solved the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 A resolution using the multiwavelength anomalous dispersion (MAD) technique. Endonuclease I exhibits strong structural specificity for four-way DNA junctions. The structure shows that it forms a symmetric homodimer arranged in two well-separated domains. Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site. While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases. T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes.

Temperature-induced changes in protein structures studied by Fourier transform infrared spectroscopy and global analysis.

Fourier transform infrared (FTIR) spectroscopy has been used to study temperature-induced structural changes which occur in albumin, immunoglobulin G, fibrinogen, lysozyme, alpha-lactalbumin, and ribonuclease S when dissolved in 2H2O. In order to analyze the data, a new method was developed in which the data were analyzed globally with the aid of a spectral model. Seven or eight bands were sufficient to fit the full data set of spectra ranging from 1420 to 1760 cm-1 with a root mean square error of 1-2% of the maximum. Subsequently, the estimated band amplitude curves which showed a sigmoidal progression with increasing temperature were (globally) fitted with a two-state thermodynamic model. In this way, information on structural changes as well as on the thermal stability of the proteins was obtained. In all proteins investigated, enhanced 1H-2H exchange occurred at temperatures well below the unfolding of the secondary structure. This was interpreted as a change in tertiary structure leading to enhanced solvent accessibility. In all the proteins investigated, except for ribonuclease S, an intermolecular beta-sheet band indicative of aggregation appeared concomitant with the denaturation of the secondary structure. The results are compared with data from other techniques and discussed in terms of local unfolding and folding intermediates.

A comparison of infrared spectra of proteins in solution and crystalline forms.

Fourier transform infrared spectroscopy has been used to compare the structure of a range of proteins in solution and in the form of single crystals. An infrared microscope was used to record the spectra of single crystals of the proteins. The proteins studied in this way were hen egg white lysozyme, bovine pancreatic ribonuclease A, bovine gamma-II crystallin, human serum amyloid P component, Endothia parasitica pepsin and Mucor pusillus pepsin. The amide I and amide II bands in the FTIR spectra of these proteins were analysed using derivative procedures thereby providing information on the secondary structure. The crystals were held under a vapour of mother liquor to reduce the effects of dehydration. A comparison of the spectra revealed that spectra recorded from crystals of lysozyme, ribonuclease A and gamma-II crystallin are nearly identical to those recorded from the proteins in solution. However, differences are observed between the spectra of serum amyloid P component, Endothia parasitica pepsin and Mucor pusillus pepsin in solution compared with that of the crystalline form These differences are suggested to be due to rearrangements of turn structures within the protein structure.

Structure and thermal stability of the extracellular fragment of human transferrin receptor at extracellular and endosomal pH.

Fourier transform infrared spectroscopy has been used to study the solution structure and thermal stability of the extracellular fragment of human transferrin receptor (tfRt) at extracellular and endosomal pH. At extracellular pH tfRt is composed of 56% alpha-helix, 19% beta-sheet and 14% turns. Upon acidification to endosomal pH the alpha-helical content of the protein is reduced and the beta-sheet content increased by nearly 10%. At extracellular pH, the midpoint temperature of thermal denaturation (Tm) for the loss of secondary and tertiary structure, and the formation of aggregated structures, is 71 degrees C. At endosomal pH this temperature is reduced by approximately 15 degrees C. The apparent entropies of thermal denaturation indicate that the native structure of tfRt at endosomal pH is far more flexible than at extracellular pH.

Fourier transform infrared spectroscopy and differential scanning calorimetry of transferrins: human serum transferrin, rabbit serum transferrin and human lactoferrin.

Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) have been used to investigate the solution structure and thermal stability of human serum transferrin (HST), human lactoferrin (HLF) and rabbit serum transferrin (RST) in their diferric and apo forms. Our study shows that: (A) The secondary structure of all the proteins studied (estimated in H2O) was in the range 43-53% alpha-helix and 23-28% beta-sheet. These values differ markedly from previously reported circular dichroism (CD) data. This is attributed to the fact that FTIR and CD measure different aspects of secondary structure (hydrogen bonding and dihedral angles, respectively). (B) The secondary structural content of the proteins is not altered by iron binding or release. However, the iron-free proteins undergo a greater extent of 1H-2H exchange than the diferric proteins indicating that significant structural changes do occur upon iron binding/release. (C) The removal of iron leads to thermal destabilization of HST, HLF and RST. Structural variation in the apo transferrins is indicated by the observation of a single irreversible DSC transition for apo human lactoferrin, a double DSC transition for apo human serum transferrin (one reversible) and a broad irreversible asymmetric DSC transition for apo rabbit serum transferrin. FTIR spectroscopy shows that a distinct loss of protein secondary structure occurs at the transition temperatures shown by DSC.