Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.     

Structural heterogeneity in waxy-rice starch

Alpha-amylase of B. amyloliquefaciens was used for the structural characterization of the amylopectin from waxy-rice starch. Fractions of -dextrins with a degree of polymerization (d.p.) <5000 were isolated from amylopectin hydrolysates after 1 and 3 h. φ,β-Limit dextrins were prepared by successive phosphorolysis and beta-amylolysis of the fractions and these were analysed by a second alpha-amylolysis. Based on the hydrolysis pattern, the limit dextrins were divided into two major groups, A and B, which possessed units of clusters of d.p. 100–200 and 90–130, respectively. An extensive alpha-amylolysis resulted in characteristic distributions of dextrins with d.p. <80 which represented branched building blocks. Type A dextrins possessed more larger building blocks with d.p. 40, but less intermediate and small blocks, than type B. The φ,β-limit dextrin of the original amylopectin had a distinct distribution enriched in small building blocks. A model is proposed in which the two types of dextrins originate from regular and less regular structural domains of the amylopectin fraction within the starch granules.     

Structural heterogeneity in intramolecular DNA triple helices

Oligodeoxynucleotides designed to form intramolecular triple helices are widely used as model systems in thermodynamic and structural studies. We now report results from UV, Raman and NMR experiments demonstrating that the strand polarity, which also determines the orientation of the connecting loops, has a considerable impact on the formation and stability of pyr x pur x pyr triple helices. There are two types of monomolecular triplexes that can be defined by the location of their purine tract at either the 5'- or 3'-end of the sequence. We have examined four pairs of oligonucleotides with the same base composition but with reversed polarity that can fold into intramolecular triple helices with seven base triplets and two T4 loops under appropriate conditions. UV spectroscopic monitoring of thermal denaturation indicates a consistently higher thermal stability for the 5'-sequences at pH 5.0 in the absence of Mg2+ ions. Raman spectra provide evidence for the formation of triple helices at pH 5 for oligomers with purine tracts located at either the 5'- or 3'-end of the sequence. However, NMR measurements reveal considerable differences in the secondary structures formed by the two types of oligonucleotides. Thus, at acidic pH significant structural heterogeneity is observed for the 3'-sequences. Employing selectively 15N-labeled oligomers, NMR experiments indicate a folding pattern for the competing structures that at least partially changes both Hoogsteen and Watson-Crick base-base interactions.     

Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin

Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.     

Structural and functional heterogeneity in an insect muscle.

Singing muscles of the katydid, Neoconocephalus robustus (Insecta, Tettigoniidae) are neurogenic, yet perform at contraction-relaxation frequencies as high as 212 Hz (Josephson and Halverson, '71). The mechanical and electrical responses of different bands of one of these muscles (the dorsal longitudinal muscle, DLM) has been examined with respect to ultrastructural features of each part which may be related to muscle performance. The DLM is composed of three bands and is innervated by four motoneurones. The cell bodies of three of these motoneurones occur ipsilaterally in the prothroracic ganglion; the cell body of the other motoneurone is contralateral in the mesothoracic ganglion. Three of the motoneurones (as yet unidentified fast axons) initiate extraordinarily fast twitches (rise time equal 7.3 msec, half duration equals 14.3 msec, 25 C), the fourth (an unidentified slower axon) evokes twitches which are considerably slower (rise time equals 18.9 msec, half duration equals 5.10 msec). Whereas the ventral and medial bands of the muscle are innervated only by fast axons (some fibers of the medial band are doubly innervated), the dorsal band is innervated by both a fast axon and the slower axon. A few fibers of the dorsal band are doubly innervated. The structure of fibers from the ventral and medial bands is very similar, with short sarcomeres (4.0 and 4.3 mum, respectively) and thin strap-like myofibrils delineated by well-developed sarcoplasmic reticulum (SR). Twenty-four percent of the volume of ventral band fibers is SR and the diffusion distance from SR to the center of the adjacent myofibril averages 0.083 mum. Twenty percent of the medial band fiber volume is SR, with a diffusion distance of 0.118 mum. Ventral and medial band fibers contain about 40% mitochondria, and 33% myofibrils. The dorsal band fibers have longer sarcomeres (9.5 mum), and only 10% of the fiber volume is SR. The muscle fibrils of the dorsal band are larger and consequently the diffusion distance is greater (0.227 mum) than in the ventral and medial bands. Mitochondria comprise 23% of the volume of dorsal band fibers. Most dorsal band mitochondria are aggregated into distinct clumps. Although some dorsal band fibers are innervated by a fast axon and some by the slower axon, the dorsal band fibers are structurally homogeneous, suggesting that neurotrophic effects are not important in maintaining the structure of dorsal band fibers. The mechanical-electrical performance and ultrastructure of the ventral and medial bands suggest their roll as fast, metabolically active but weak muscles, used in singing; the dorsal band as a slower but stronger muscle, perhaps involved in postural movements of the wing during singing.     

Structural heterogeneity of terminal glycans in Campylobacter jejuni lipooligosaccharides

Lipooligosaccharides of the gastrointestinal pathogen Campylobacter jejuni are regarded as a major virulence factor and are implicated in the production of cross-reactive antibodies against host gangliosides, which leads to the development of autoimmune neuropathies such as Guillain-Barré and Fisher Syndromes. C. jejuni strains are known to produce diverse LOS structures encoded by more than 19 types of LOS biosynthesis clusters. This study demonstrates that the final C. jejuni LOS structure cannot always be predicted from the genetic composition of the LOS biosynthesis cluster, as determined by novel lectin array analysis of the terminal LOS glycans. The differences were shown to be partially facilitated by the differential on/off status of three genes wlaN, cst and cj1144-45. The on/off status of these genes was also analysed in C. jejuni strains grown in vitro and in vivo, isolated directly from the host animal without passaging, using immunoseparation. Importantly, C. jejuni strains 331, 421 and 520 encoding cluster type C were shown to produce different LOS, mimicking asialo GM(1), asialo GM(2) and a heterogeneous mix of gangliosides and other glycoconjugates respectively. In addition, individual C. jejuni colonies were shown to consistently produce heterogeneous LOS structures, irrespective of the cluster type and the status of phase variable genes. Furthermore we describe C. jejuni strains (351 and 375) with LOS clusters that do not match any of the previously described LOS clusters, yet are able to produce LOS with asialo GM(2)-like mimicries. The LOS biosynthesis clusters of these strains are likely to contain genes that code for LOS biosynthesis machinery previously not identified, yet capable of synthesising LOS mimicking gangliosides.     

Nucleation of protein crystals

This paper introduces nucleation theory applied to crystallizing protein solutions. It is shown that the classical approach explains the available nucleation data under most conditions used for growing protein crystals for structural studies and for industrial crystallization. However, it fails to explain most experimental data on the structure of the critical clusters. It is also shown that for open systems working out of equilibrium, such as hanging-drop and counterdiffusion techniques, the geometry of the Ostwald-Myers protein solubility diagram and the number, size, and quality of the forming crystals depend not only on supersaturation but also on the rate of development of supersaturation.     

Structural heterogeneity of the lipopolysaccharides of the Neisseriaceae

Lipopolysaccharides from 5 different genera of the Neisseriaceae were analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and visualized by silver staining. Significant heterogeneity in the banding patterns was observed with some of the strains producing only low molecular mass molecules and others producing O-repeating units. All genera examined except Branhamella contained strains that were able to produce an O-repeating side chain on their lipopolysaccharides. The ability to produce the repeating subunit did not correlate with the presence of plasmids.     

Conformational interconversion in protein crystals.

We present evidence that the structure of carbonmonoxy myoglobin crystals can be altered by lowering the pH. This structural change is monitored by the characteristic Fe-CO Raman modes at 508 and 491 cm-1 and is thought to involve a localized distal pocket transition from a "closed" conformation at pH 7 to a more "open" conformation at pH 4. These changes take place in the crystal without loss of intensity of a conformationally sensitive Raman mode at 252 cm-1 that signals a partial unfolding of the globin structure in solution. Quantitative studies, which monitor the open and closed populations as a function of laser photolysis, demonstrate that the interconversion rates (k+/-) in solution at 298 K are fast compared to the photolysis and CO entry rates (i.e. k+/- much greater than 10(3) s-1), while in frozen samples the interconversion is much slower than the experimental time scale (minutes). Since the open conformation is a minority species at pH 7, rapid exchange in aqueous solution is a necessary condition for this species to play a functional role. In the crystal, the interconversion rates are slowed compared to solution and begin to approach the photolysis rate (i.e. k+/- approximately 10(3) to 10(4) s-1). This indicates that the barriers for conformational exchange are increased in the crystal environment, compared to the solution, apparently due to the packing forces of the surrounding molecules. X-ray and neutron diffraction studies of MbCO crystals at high and low pH are needed to characterize the details of the structural changes and to test the hypothesis that closed and open distal pocket structures are associated with the 508 and 491 cm-1 Fe-CO modes.     

Structural heterogeneity regarding local Shwartzman activity of lipid A

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin-layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515--Ec-A2 and Ec-A3--exhibited strong activity, similar to that of previous synthetic E. coli-type lipid A (compound 506 or LA-15-PP). The Ec-A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec-A2 fraction was structurally similar to compound 506 except that it carried a 3-hydroxytetradecanoyl group at the C-3' position of the backbone in place of a 3-tetradecanoyloxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec-A2 (12 C) and Ec-A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec-A2 or Ec-A3. In these preparations, a large part of the 3-dodecanoyloxytetradecanoyl group might be replaced by 3-hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella-type lipid A (compound 516 or LA-16-PP) exhibited low activity. A lipid A fraction, Cv-A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C-2 and C-2' positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester, and these lipid A (Pd-A3 and Pv-A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.     

Structural heterogeneity and genomic distribution of Drosophila melanogaster LTR-retrotransposons

Structural heterogeneity of five long terminal repeat (LTR) retrotransposon families (297, mdg 1, 412, copia, and 1731) was investigated in Drosophila melanogaster. The genomic distribution of canonical and rearranged elements was studied by comparing hybridization patterns of Southern blots on salivary glands from adult females and males with in situ hybridization on polytene chromosomes. The proportion and genomic distribution of noncanonical copies is distinctive to each family and presents constant features in the four different D. melanogaster strains studied. Most elements of families 297 and mdg 1 were noncanonical and presented large interstock and intrastock polymorphism. Noncanonical elements of these two families were mostly located in euchromatin, although not restricted to it. The elements of families 412 and copia were better conserved. The proportion of noncanonical elements was lower. The 1731 family is mainly composed of noncanonical, beta-heterochromatic elements that are highly conserved among stocks. The relation of structural polymorphism to phylogeny, transpositional activity and the role of natural selection in the maintenance of transposable elements are discussed.     

Specific and non-specific contacts in protein crystals

Statistical analysis of protein-protein interfaces in a database of pure peptide crystals shows that the distribution of the contact area contains two components: a major exponential distribution and a minor flat distribution. Analysis of two sub-databases provides evidence that the two components represent specific and non-specific contacts, respectively. The probability of an interface with a given area being specific can be estimated. A scaled quantity (contact ratio) is introduced that is more useful than contact area for discriminating specific and non-specific contacts in protein crystals.     

Structural heterogeneity of wheat arabinoxylans revealed by Raman spectroscopy

A set of arabinoxylan samples differing in their arabinose composition and various samples of arabino-xylo-oligosaccharide samples were analysed by Raman spectroscopy. Specific signatures for arabinose substitution were found in several spectral regions, that is, 400-600, 800-950 and 1030-1100 cm(-1). A linear relationship was observed between the peak ratio 855/895 cm(-1) of the second derivative spectra and the A/X ratio determined by chemical analysis. Moreover, spectral changes were observed in the 400-600 cm(-1) region assigned to the coupled vibrations mode in the skeleton: while the intensity of the band at 570 cm(-1) increased with the degree of substitution, that at 494 cm(-1) decreased. Similarly, a linear relationship was observed between the peak intensity ratio 570/494 cm(-1) calculated on the second derivative spectra and the composition data. Analysis of Raman spectra of arabino-xylo-oligosaccharides allowed to identify specific spectral features of disubstitution.     

Structural heterogeneity of the basement membrane in the rat proximal tubule

Summary The ultrastructure of the basement membrane of the rat proximal tubule was observed by transmission electron microscopy after the use of a cold dehydration technique. The basement membrane of the P1 segment is thick and possesses several structural specializations that are rare in other basement membranes; these include intraepithelial ridges, dense bars, and basement membrane vesicles. The intraepithelial ridges are found in the intercellular spaces between interdigitating processes of the proximal tubule cells. The ridges and the interdigitating processes run circumferentially around the tubule. The dense bars are frequently found in the intraepithelial ridges. They are especially prominent on the concave side of the tubular bends and to a lesser extent near sites where intracellular actin filaments anchor onto the basal cell membranes. The basement membrane vesicles are bounded by unit membranes; they are variable in both their electron density and their size. They are usually found in association with dense bars, and the grade of their accumulation is positively correlated with the development of the dense bars. These three specializations have no topographical relationship with the interstitial structures, such as fibrobalasts and collagen fibrils. The specializations are best developed on the concave side of tubular bends where the circumferential stresses caused by the intraluminal hydraulic pressure are presumably the largest; we therefore propose that they are an adaptation to, or a manifestation of, the increased wall stress in the proximal tubule.     

Sindbis virus core protein crystals

The core protein of Sindbis virus has been crystallized. Three different crystal forms have been observed. They diffract variously from 2.5 A to 3.5 A resolution.     

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.