Predicted secondary and tertiary structures of carp gamma-crystallins with high methionine content: role of methionine residues in the protein stability.

A systematic structural comparison of several carp gamma-crystallins with high methionine contents was made by the secondary-structure prediction together with computer model-building based on the established X-ray structure of calf gamma-II crystallin. The overall surface hydrophilicity profile and the distribution of helices, beta-sheets, and beta-turns along the polypeptide chains are very similar among these carp gamma-crystallins. In addition, their general polypeptide packing is close to the characteristic 2 domain/4 motif Greek key three-dimensional conformation depicted for the calf gamma-II crystallin. Interestingly, most hydrophobic methionine residues are located on the protein surface with only a few buried inside the protein surface or in the interface between two motifs of each domain. The exposed hydrophobic and polarizable methionine cluster on the protein surface may have a bearing on the crystallin stability and dense packing in the piscine species, and probably also provides a malleable nonpolar surface for the interaction with other crystallin components for the maintenance of a clear and transparent lens.     

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

Carp gamma-crystallins with high methionine content: cloning and sequencing of the complementary DNA

The nucleotide sequences of gamma-crystallin cDNAs cloned from the common carp (Cyprinus carpio) have been determined. The amino-acid sequences derived consist of two polypeptides with 177 and 172 amino-acid residues for gamma-m1 and gamma-m2, respectively. They exhibit unusually high methionine contents: 12.4% for gamma-m1 and 14% for gamma-m2. Comparison of both fish gamma-crystallins with bovine gamma-II crystallin reveals that they are similar in structure. The striking features of both fish gamma-crystallins are as follows. (1) Both of them retain the 'conserved' residues, i.e., Tyr-6, Glu-7, Gly-13, Ser-34 and their equivalents in other motifs. (2) they possess the second aromatic residue at position 11. Both of these structural features are considered to be the major factors in stabilizing the folded hairpin structure of the protein. (3) The variable residues in the core region of C-terminal domain are almost all sulfur-containing amino acids, i.e., methionine or cysteine. (4) 30% of the surface hydrophobic groups are composed of methionine. The last two unusual features have been found so far only in these two fish gamma-crystallins. The high methionine content may make an important contribution to the protein stability of fish gamma-crystallins.     

A computer graphics model of frog gamma-crystallin based on the three-dimensional structure of calf gamma-II crystallin

Molecular models for Rana gamma-1 and gamma-2 crystallins have been constructed using computer graphics on the basis of the protein primary structure derived from the complementary DNA sequence and the three-dimensional structure of calf gamma-II crystallin that has been defined at high resolution by X-ray analysis. The models show that the cores of the two domains are conserved as hydrophobic, with the polypeptide chain arranged as a four Greek-key motif structure. Although many lysines replace arginines at equivalent positions in mammalian proteins, the Rana crystallins also have an extensive series of ion pairs on their surface; these are strongly implicated in their function as stable structural molecules, which are highly conserved in the evolution of the vertebrate eye lens.     

喇曼光谱技术在生物医学中的应用

喇曼光谱技术是一种非侵入、非弹性散射技术,能够在分子层次上探测物质的临床医学特征和结构特征。本文综述了近十年来喇曼光谱技术在生物医学中的最新发展,归纳出了四种目前在生物医学中最为活跃的喇曼光谱技术：近红外喇曼光谱、紫外共振喇曼光谱、表面增强喇曼光谱和多维喇曼成像技术。详细阐述了这四种技术的特点和适用范围,并且列举了丰富的成功范例。     

Structural analysis and comparison of cobrotoxin and cardiotoxins by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. The near-IR FT-Raman analytical method has been applied to the characterization and classification of the toxin components in their lyophilized forms. Structural analysis and comparison of various purified toxin fractions were made with respect to their amino acid compositions and near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structure of cobra toxins including cobrotoxin and various cardiotoxins is mainly anti-parallel beta-pleated sheet as judged by the Raman signals at 1238 cm-1 (amide III) and 1671 cm-1 (amide I). It is also found that the relative Raman signal intensities of Tyr, Phe, Trp and Met residues in purified toxins correlate very well with the structural data obtained from amino acid analysis. The advantage and improvement of applying the near-IR FT-Raman spectroscopy to the unambiguous classification and comparison of venom toxins are evident and the discrepancies with previous Raman studies on these venom toxins are also revealed and discussed.     

Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution

The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general.     

Raman spectroscopic evidence for a disulfide bridge in calf gamma II crystallin

Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical gamma II crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500-2600 cm-1), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of gamma II as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500-540 cm-1), the native form shows a line near 511 cm-1 which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:1 computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm-1. In every other spectral region, both native and reduced gamma II forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution.     

Comparison of the gamma-crystallins isolated from eye lenses of shark and carp. Unique secondary and tertiary structure of shark gamma-crystallin

gamma-Crystallin isolated from the shark of cartilaginous fishes was compared with the cognate gamma-crystallin from the carp of bony fishes. Distinct differences in amino acid compositions, primary, secondary and tertiary structures were found. The most salient features of shark gamma-crystallin lie in the fact that this crystallin possessed a significant alpha-helical structure in the peptide backbone as revealed by circular dichroism study, in contrast to those orthologous gamma-crystallins from other vertebrate species including bony fishes which all show a predominant beta-sheet secondary structure. The tertiary structure as reflected in the intrinsic microenvironments of various aromatic amino acids in the native crystallins also shows unambiguous differences between these two classes of gamma-crystallins. N-Terminal sequence analysis corroborates the structural differences between shark and carp gamma-crystallins. gamma-Crystallin from the more primitive shark seems to be more in line with the main evolutionary phylogeny leading to the modern mammalian gamma-crystallin.     

Raman spectroscopy in chemical bioanalysis

Advances in instrumentation are making Raman spectroscopy the tool of choice for an increasing number of (bio)chemical applications. Raman is an interesting option for several reasons, including the sensitivity to small structural changes, non-invasive sampling capability, minimal sample preparation, and high spatial resolution in the case of Raman micro-spectroscopy. Herein we discuss the most recent technical approaches employed, from the well-known surface enhanced resonance Raman spectroscopy to non-linear Raman techniques such as coherent anti-stokes Raman spectroscopy (CARS) and related techniques. Relevant applications of Raman spectroscopy in the fields of clinical pathology, in vivo and ex vivo imaging, classification and detection of microorganisms and chemical analysis in the past three years are also included.     

Peptide scanning-based identification of regions of gamma-II crystallin involved in thermal aggregation: evidence of the involvement of structurally analogous,helix-containing loops from the two double Greek key domains of the molecule

Gamma crystallin is one of three structural proteins present in great abundance in the fiber cells of the vertebrate eye lens. The protein displays a tendency to aggregate readily in the course of heating, cooling, being exposed to ultraviolet radiation, or rapid refolding. To investigate the molecular mechanisms underlying such aggregation, we have employed a peptide-scanning approach aimed at identifying regions of bovine gamma-II crystallin that may be involved in intermolecular interactions leading to aggregation, using assays that measure the competitive inhibition of such aggregation by reagents drawn from a group of contiguous (overlapping) peptides derived from the sequence of the protein itself. Our results suggest that two regions, comprising residues 61-74, and 145-159, play key roles in aggregative interactions. Intriguingly, the two regions (each containing a solvent-exposed, single-turn helix in the native structure) are located in structurally analogous positions in the two homologous double Greek key (beta sheet) domains of the protein, suggesting that helix-strand conversions may operate to facilitate intermolecular beta sheet interactions during aggregation.     

Evolutionary conservatism of the molecular structure of gamma-crystallins of vertebrates

Homology of 18 amino acid sequences of lens gamma-crystallins of several vertebrates: frog, mouse, rat, calf and human being--has been considered. Pair sequence homology varies in the range from 57 to 100%, the mean value is equal to 74%. The spatial structures have been determined only for two calf gamma-crystallins. The protein molecule consists of four-fold repeated "motifs" (patterns) which are joint in two domains. After comparison of 18 gamma-crystallin sequences it was found that "motifs" domains and whole protein molecules have about 10, 30 and 58% conservative residues, respectively, that seem to be related to the evolution of these structural units. Structure analysis shows that almost all the conservative residues have an important structural meaning and play a basic role in the domain and molecular structure organization. This result allows us to make a conclusion about the homology of spatial structures of all considered gamma-crystallins of vertebrates.     

The reaction of glutathione with the eye-lens protein gamma-crystallin.

Lens cells contain high concentrations of thiol-rich proteins, gamma-crystallins and reduced glutathione. Solutions of bovine gamma-crystallin react avidly with either reduced or oxidized glutathione to form protein-glutathione mixed disulphides. A method of purification of a gamma-II crystallin-glutathione adduct containing two mixed disulphide groups is described.     

FT-Raman spectroscopy as diagnostic tool of Congo red binding to amyloids

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties protecting the oocyte and developing embryo from environmental hazards. We present data from FT-Raman spectroscopy of silkmoth chorion and amyloid-like fibrils formed from peptide analogues of chorion proteins, both unstained and stained by Congo red. The results show that FT-Raman spectroscopy is not a straightforward diagnostic tool for the specific interactions of Congo red with amyloids: a dilute aqueous solution of the Congo red dye at pH 5.5 and a thin solid film of the dye cast from this solution exhibit the same "diagnostic" Raman shifts relative to the neat Congo red dry powder as do amyloid fibrils formed from peptide analogues of chorion proteins stained by Congo red. An important consequence of this finding is that these shifts of the Raman active modes of Congo red are probably due to the formation of supramolecular dye aggregates in the presence of water. Therefore, this is not an appropriate diagnostic test for Congo red binding to amyloids.     

Polarized Raman spectra of oriented fibers of A DNA and B DNA: anisotropic and isotropic local Raman tensors of base and backbone vibrations.

Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies.     

A Raman spectroscopic study of hen egg yolk phosvitin: structures in solution and in the solid state

Laser Raman spectroscopy has been employed to study the structure of the hen egg yolk protein phosvitin in H2O and D2O solutions at neutral and acidic pH (pD) and in the solid state. The Raman data indicate an unusual conformation for phosvitin in neutral aqueous solution, which is deficient in both alpha-helix and conventional beta-sheet conformations. This unusual pH 7 structure is, however, largely converted to a beta-sheet conformation in strongly acidic media (pH less than 2). beta-Sheet is also the predominant secondary structure for phosvitin in the solid state, obtained by lyophilization of the protein from aqueous solution at neutral pH. The imidazolium rings of histidyl residues remain significantly protonated near neutrality, which suggests substantial elevation of the pK for imidazolium ring ionizations of phosvitin in aqueous solution. This may result from extensive ion-pair interactions involving positively charged histidines and negatively charged phosphoserines, which are prevalent in the phosvitin sequence. The present results suggest that antiparallel beta-sheets may not be the secondary structure most characteristic of native phosvitin (physiological pH), even though beta-sheet is the predominant conformation for phosvitin in acidic solutions (pH 1.5) and in the lyophilized solid. Phosvitin appears to be the first protein for which the major component to the Raman amide I band is centered near 1685 cm-1, which is 10-40 cm-1 higher than proteins heretofore examined in aqueous solution by Raman spectroscopy.     

Physicochemical characterization of lens crystallins from the carp and biochemical comparison with other vertebrate and invertebrate crystallins

Lens crystallins were isolated from the homogenate of carp (Cyprinus carpio) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, immunodiffusion, amino acid analysis, circular dichroism, and protein sequence analysis. Three well-defined fractions corresponding to alpha/beta-, beta-, and gamma-crystallins were obtained in relative weight percentages of 26, 22, and 52%. The native molecular masses of the purified fractions were determined to be 410, 60, and 20 kDa, respectively. The polypeptide compositions as determined by SDS gel electrophoresis revealed the substantial presence of beta-crystallin polypeptides in the alpha-crystallin fraction; this is also evident in the fractionation of amphibian crystallins but is not common in the case of higher classes of vertebrates. The circular dichroism spectra indicate a predominant beta-sheet structure in all three fractions, albeit with some contribution of alpha-helical structure in the gamma-crystallin, the amino acid composition of which bears a resemblance to that of squid crystallin. Sequence comparison of carp gamma-crystallin with frog and calf gamma-crystallins indicates a high degree of homology in their N-terminal segments despite the dissimilarity of amino acid compositions and weak immunological cross-reactivity.     

Structure and stability of gamma-crystallins: tryptophan, tyrosine, and cysteine accessibility

The solute perturbation techniques of fluorescence of tryptophan (Trp) and dye-labeled thiol groups of cysteine as well as phosphorescence of tyrosine (Tyr) were utilized to obtain information on the relative solvent exposure and accessibility of these residues in gamma-crystallins. Both acrylamide and iodide quenchers were used to evaluate the quenching parameters in terms of accessibility and charge characteristics of the proteins. Stern-Volmer plots reveal the presence of more than one class of Trp residues in gamma-III and gamma-IV, and these residues in gamma-II are least accessible compared to the other two. Both steady-state and lifetime quenching studies of the dye-labeled fluorescence indicate that distinct differences also exist among these crystallins in cysteine (Cys) accessibilities. All three proteins, gamma-II, gamma-III, and gamma-IV, show two distinct lifetime components of the dye-labeled Cys residues. Both components of gamma-II undergo dynamic quenching, whereas only the major component of the other two crystallins is affected by the quenchers. Addition of acrylamide causes a decrease in Tyr phosphorescence of gamma-III and gamma-IV, but no change in the emission of gamma-II. The decrease is attributed to the formation of a nonemittive ground-state complex between the acrylamide and Tyr of the proteins; the association constant, Ka, calculated from the emission data, has been considered as a measure of Tyr accessibility. Ka values indicate that Tyr residues in gamma-III are most exposed and accessible compared to those in the other two proteins. Results of quenching by iodide ion reveal significant differences in the surface charge of the proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.