Infrared absorption spectrum of keratin. I. Spectra of alpha-, beta-, and supercontracted keratin

A number of basic features of the infrared spectrum of keratin have been confirmed and some new features have been found. In the 3-μ region, the amide A frequency of helical material in α-keratin at 3286 cm.^?1 is close to the expected value, but that of the crystalline phase in α-keratin, near 3270 cm.^?1, is lower than had previously been reported. The noncrystalline phase absorbs in the vicinity of 3300 cm.^?1 or above, and this causes the low-intensity component of the amide A band in both α- and β-keratin to occur at higher frequencies than those of the high-intensity component. In the 6-μ region, the amide II frequency of noncrystalline material is below 1525 cm.^?1. Keratin denatured in lithium bromide, after washing out the reagent, appears to have a considerable helix content, possibly as much as that of the original protein. Hydration causes significant spectral changes. In the 6-mu; region, the frequency of the amide I band of crystalline material is lowered, while that of the amide II band is increased, both by a few wavenumbers; the amide II frequency of noncrystaline material is also increased by a few wavenumbers. In the 3-μ region, no significant change is observed in the amide A frequency of crystalline material, while the frequency of the noncrystaline material is reduced. These spectral changes are interpreted in terms of a weak association of water with main-chain carbonyl groups in the crystalline phase, while in the noncrystaline phase it is thought likely that water molecules form hydrogen-bond bridges between polypetide chains. The absorption coefficient of the amide A band and the integrated absorption intensities of the amide A, I, and II bands do not vary appreciably in the three forms of keratin investigated.     

Infrared absorption spectrum of keratin. II. Deuteration studies

A number of new bands have been found in the spectra of deuterated α- and β-keratin. In particular, the deuteration difference spectrum has been useful for the determination of frequencies of previously unsuspected bands. Thus it is found that the amide A and II frequencies of the nonhelical component in α-keratin occur at 3310–3320 and 1520 cm.^?1, respectively, and that both bands exhibit dichroism consistent with polypeptide chains which have a measure of alignment parallel to the fiber axis. The parallel dichroism of the amide II′ band of this phase at about 1435 cm.^?l also indicates some alignment. A nondichroic residual band at 1513 cm.^?1 in highly deuterated α-keratin is assigned to the tyrosine residue, as a sharp band near this frequency is found in the spectrum of polytyrosine. The ν‖(o) component of the α-helix is weak or absent in α-keratin, and the relatively sharp band observed near this frequency is thought to be due to the tyrosine residue, while its dichroism is caused by the presence of dichroic nonhelical material. A band near 1575 cm.^?1 in deuterated α- and β-keratin is tentatively assigned to the deuterated guanidinium group of arginine. This band becomes progressively more prominent during deuteration, which indicates that some arginine side chains arc slow to exchange, possibly because their environment prevents interaction with D₂O. The deuteration difference spectrum also shows that, contrary to earlier views, helical material in α-keratin exchanges significantly during the early stages of deuteration, although at a slower rate than the nonhelical material, while part of the nonhelical phase does not exchange as rapidly as had been thought and makes a contribution even after many hours or days.     

X-ray diffraction and infrared studies of an -helical fragment from -keratin

Fragments of α-keratin were obtained by partial proteolysis of the low-sulphur protein fraction, S-carboxymethylkerateine-A, from wool. X-ray diffraction and infrared absorption studies were made on oriented films formed from these fragments. The results indicate that the fragments have a coiled-coil α-helical conformation similar to that in α-keratins, and that the films consist of particles approximately 160 Å in length arranged end-to-end.     

β-structure from the microfibillar proteins of Merino wool

The β-structure of S-caboxymethyl derivatives of microfibrillar proteins isolated from Merino wool was investigated by X-ray diffraction for comparison with the structur of β-keratin. The S-carboxymethylated microfibrillar proteins(SCMKA) w well-oriented β-films of SCMKA weer obtained by stretching the SCMKA cast films in steam up to about 300% extesnsion. It was found that the reflections in β-pattern of SCMKA may be indexed on a pseudo-orthorhombic unit cell with a =0.94 nm, b = 0.66 nm and c = nm, where the ab, and c axes are in the direction of the interchain hydrogen bonding, the main chain(fibre axis) and the side chain, respectively. The unit cell dimesnions evaluated for SCMKA were almost the same as those for β-keratin, suggeting that few peptide sequences containing S-carboxymethyl cystine may be involved in the formation of β-structure from SCMKA.     

Development of an eco-friendly approach for biogenesis of silver nanoparticles using spores of Bacillus athrophaeus

The biological synthesis methods have been emerging as a promising new approach for production of nanoparticles due to their simplicity and non-toxicity. In the present study, spores of Bacillus athrophaeus were used to achieve the objective of developing a green synthesis method of silver nanoparticles. Enzyme assay revealed that the spores and their heat inactivated forms (microcapsules) were highly active and their enzymatic contents differed from the vegetative cells. Laccase, glucose oxidase, and alkaline phosphatase activities were detected in the dormant forms, but not in the vegetative cells. Although no nanoparticle was produced by active cells of B. athrophaeus, both spores and microcapsules were efficiently capable of reducing the silver ions (Ag⁺) to elemental silver (Ag⁰) leading to the formation of nanoparticles from silver nitrate (AgNO₃). The presence of biologically synthesized silver nanoparticles was determined by obtaining broad spectra with maximum absorbance at 400 nm in UV–visible spectroscopy. The X-ray diffraction analysis pattern revealed that the nanoscale particles have crystalline nature with various topologies, as confirmed by transmission electron microscopy (TEM). The TEM micrograph showed the nanocrystal structures with dimensions ranging from 5 to 30 nm. Accordingly, the spore mixture could be employed as a factory for detoxification of heavy metals and subsequent production of nanoparticles. This research introduces an environmental friendly and cost effective biotechnological process for the extracellular synthesis of silver nanoparticles using the bacterial spores.     

On the rigidity of RNA in tomato bushy stunt virus

The motional state of RNA in tomato bushy stunt virus, both in the crystalline state and in solution, has been investigated using ³¹P nuclear magnetic resonance methods. It has been found that the RNA is highly immobile in the native virus and it is suggested that the lack of a high-resolution X-ray diffraction pattern for either the RNA or the N-terminal regions of the protein coat molecules (Harrison et al., 1978) is due to static disorder in the crystals. Dynamic disorder has been detected in the virus after treatment with EDTA, which causes a structural change and an increase in particle size.     

Structure of intermediate filaments

Recent amino acid sequence data have revealed that the microfibrils in hard α-keratin contain proteins with highly significant homologies and closely similar structural characteristics to the intermediate filament (IF) proteins known as desmin and vimentin. This result implies that microfibrils in hard α-keratin may be classified as a member of the IF and that the major features of these various filamentous structures are the same. Consequently, data obtained using X-ray diffraction, electron microscopy, amino acid sequence structural analysis and physicochemical techniques have been collated from the hitherto diverse fields of keratin and IF structure and used to formulate a more detailed model for the 7–8 nm diameter filaments than has previously been possible. Two models consisting of four-chain units arranged with the helical symmetry deduced for hard α-keratin¹ (Fraser et al. J. Mol. Biol. 1976, 108, 435–452) are in accord with the data. The structural unit comprises an oppositely directed pair of molecules each consisting of a two-stranded parallel-chain coiled-coil rope of length ～45 nm stabilized by both interchain and intermolecular ionic interactions. For a perfectly regular structure the filament may be likened either to a seven-stranded cable with a supercoil pitch length of about 345 nm (pitch angle ～2.9°), or a ten-stranded cable (Fraser, R. D. B. and MacRae, T. P. Polymer 1973, 14, 61–67) with a supercoil pitch length of about 1293 nm (pitch angle ～0.8°). The models also provide some insight into the self-assembly mechanism of the IF.     

Lepidosaur ß-keratin chains with four 34-residue repeats: Modelling reveals a potential filament-crosslinking role

ß-keratin chains contain a characteristic and homologous 34-residue sequence, which is believed to adopt a twisted ß-sheet conformation that assembles in an antiparallel manner with a similar sheet in a second chain to form a ß-sandwich. These sandwiches are, in turn, related to one another by a left-handed four-fold screw axis to generate a helical structure that forms the core of the 3.4 nm diameter filaments observed by electron microscopy and deduced from X-ray fibre diffraction. Recently, it has been shown that one ß-keratin chain, with a molecular weight approximately twice that of the majority of ß-keratin chains, is conserved across the lepidosaurs (lizards, snakes and tuatara). Uniquely, it contains four 34-residue repeats. Although this chain is a minor component the observation that the entire chain shows a high degree of sequence conservation between species suggests an important structural/functional role in vivo. Modelling shows that only six families of structures are physically possible. In three of these the repeats exist within a single filament and might therefore act in a filament nucleation role. In the second three families the repeats exist in two, three or four filaments, implying that their function may be to act as an inter-filament crosslinker, thereby providing lateral reinforcement to the epidermal appendage. The favoured model is one in which the first two repeats form a β-sandwich in one filament and the second two repeats form a β-sandwich in a neighbouring filament. Links between alternating up- and down-pointing β-sheets would provide optimum connectivity.     

Coiled coil formation and sequence regularities in the helical regions of α-keratin

Models of the helical portions of wool α-keratin sequenced by Crewther et al. (1976) show that they build well into a parallel two-stranded rope but three chains are unlikely to fit into any three-stranded rope. The 9.5-residue periodic pattern in the charged residues discovered by Parry et al. (1977) has been analysed further, suggesting that 9.5 rather than 28 residues is the fundamental period. The positively charged amino acids segregate into 11 zones, 3.5 residues wide and 9.5 residues apart. Possible structures for clusters of two-stranded coils are considered but none appear able to account for a strong 9.5-residue period. Instead it is probable that the pattern is related to a periodic interaction between the helical and non-helical portions of α-keratin.     

Architecture and Assembly of the Bacillus subtilis Spore Coat

Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization pattern in a biological organism.     

Disordered hydrogen bonding in N-(1-deoxy-β-d-fructopyranos-1-yl)-N-allylaniline

We report on a ¹³C NMR and a single-crystal X-ray diffraction study of N-(1-deoxy-β-d-fructopyranos-1-yl)-N-allylaniline (d-fructose-N-allylaniline). In solution, an equilibrium of α-pyranose, β-pyranose, α-furanose, β-furanose, and acyclic keto tautomers of the carbohydrate was detected in the following respective proportions: 2.2%, 47.4%, 4.5%, 33.6%, and 12.3%. In the crystalline state, the compound exists exclusively as the β-pyranose form, in the normal ²C₅ chair conformation. Bond lengths and valence angles compare well with the average values from a number of β-fructopyranose derivatives. The structure displays two unusual features for this class of compounds. First, the molecule assumes an eclipsed conformation around the C1-C2 bond, apparently stabilized by an intramolecular O2-H···N hydrogen bond. Second, the O3, O4, and O5 hydroxyl groups are involved in an intermolecular hydrogen bonding, which forms 12-membered homodromic cycles. In the cycles, each determined hydrogen atom site is half occupied, possibly due to the ···H-O···H-O··· ? ···O-H···O-H··· flip-flop type disorder.     

Studies of glucosidase activities from surface sediments in mangrove swamp

Four transects including sixteen stations were established in the Fugong mangrove (117°54′-117°55′E, 24°22′-24°24′N) of the Jiulong River Estuary, Fujian, China. Besides geochemical characterization and estimation of bacterial abundances, the distribution of α- and β-glucosidase activity was studied to explore the degradation of carbohydrates which can be expected to occur in high quantities in mangrove systems. The distribution pattern of microbial α-glucosidase and β-glucosidase activities was investigated using a fluorogenic model substrate (FMS) technique in order to allow better understanding of in situ enzyme activities, as well as their relation to bacterial biomass, metabolic activity and environmental factors in mangrove sediments. The results showed that the enzyme activities of α-glucosidase (10.83~100.86 µmol g^- 1 h^- 1) and β-glucosidase (39.60~222.75 µmol g^- 1 h^- 1) varied among the different stations, and the enzyme activities of β-glucosidase were higher than those of α-glucosidase at all stations. The extracellular enzyme activities were positively related to organic C, organic matter and bacterial abundance. In addition, the use of the FMS technique to measure extracellular enzyme activities of mangrove sediments could help us to evaluate their catabolic behavior in situ and so lead to a better understanding of the bacterial role in material cycle of mangrove swamp ecosystems.     

Investigation of spore coat display of Bacillus subtilis β-galactosidase for developing of whole cell biocatalyst

The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses β-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101(ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of β-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction’s mixture.     

The crystal structure of 6-epi-desacetyllaurenobiolide,a germacra-1(10),4-diene-12,8α-olide from Montanoa grandiflora

A chloroform extract of Montanoa grandiflora afforded a novel 6β-hydroxy-germacradien-8,12-olide. Its structure was shown to be 6-epi-desacetyllaurenobiolide by spectral studies, chemical transformations and single crystal X-ray diffraction. The X-ray data demonstrate that the ten-membered ring exists in the crystal in the highly unusual [₁₅D⁵,₁D¹⁴] conformation, in which the methyl group at C-4 is α-oriented and the methyl group at C-10 is β-oriented. The two double bonds are approximately parallel rather than crossed.     

A simple preparation of beta-trypsin based on a calorimetric study of the thermal stabilities of alpha- and beta-trypsin

Bovine trypsin preparations contain, in addition to the single chain form of the enzyme, an active two-chain autolysis product (Schroeder, D. D., and Shaw, E., J. Biol. Chem. (1968), 243, 2943–2949). Differential scanning calorimetric (DSC) studies showed that the single chain form, β-trypsin, is more stable to thermal denaturation than the two-chain form, α-trypsin. Rate constants and activation energies for the thermal denaturation of β-trypsin are 5 × 10^?5 sec^?1 and 69 kcal/mole and of α-trypsin are 5 × 10^?3 sec^?1 and 38 kcal/mole at pH 4.4 and 48 °C. Preparation of pure β-trypsin can be greatly simplified by prior thermal denaturation of the α form. At least 75% of the α form is denatured by heating a 10–15% solution of commercial crystalline trypsin for 30–45 min at 48 °C, pH 4.4, 0.02 m Ca²⁺. The native β-trypsin is then easily isolated from the denatured α-trypsin by batchwise adsorption onto ovoinhibitor-agarose at pH 8. After elution at pH 2, dialysis, and lyophilization an average preparation contained approximately 85% β-trypsin, 10% α-trypsin, and 5% inactive material. Benzamidine was used during the isolation to decrease the rate of conversion of β- to α-trypsin. Because the separation of active β-trypsin from heat-denatured α-trypsin is relatively easy, the total preparation time has been reduced to 1 day.     

A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity.

α-Amylases have been found to convert starch and glycogen, in part, to products other than hemiacetal-bearing entities (maltose, maltodextrins, etc.)—hitherto, the only products obtained from natural α-glucans by α-amylolysis. Glycosides of maltosaccharides were synthesized by purified α-amylases acting on starch or bacterial glycogen in the presence of p-nitrophenyl α- or β-d-glucoside. From a digest with crystallized B. subtilis var. amyloliquefaciens α-amylase, containing 4 mg/ml of [¹⁴C]glycogen and 40 mmp-NP β-d-glucoside, three pairs of correspondingly labeled glycosides and sugars were recovered: p-NP α-d-[¹⁴C]glucopyranosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]glucose; p-NP α-[¹⁴C]maltosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltose; p-NP α-[¹⁴C]maltotriosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltotriose. The three glycosides accounted for 11.4% of the [¹⁴C]glycogen donor substrate; the three comparable sugars, for 30.4%; higher maltodextrins, for 58.2%. Calculations based on the molar yields of all reaction products show that [¹⁴C]glycosyl moieties were transferred from donor to p-NP β-d-glucoside with a frequency of 0.234 relative to all transfers to water. This is a very high value considering the minute molar ratio (0.0007) of β-d-glucoside-to-water concentration. Less striking but similar findings were obtained with cryst. hog pancreatic and Aspergillus oryzae α-amylases. The results extend earlier findings (Hehre et al., Advan. Chem. Ser. (1973) 117, 309) in showing that α-amylases have a substantial capacity to utilize the C₄-carbinols of certain d-glucosyl compounds as acceptor sites.     

The fine structure of silk fibroin

The fine structure of Bombyx mori silk fibroin was investigated by electron microscopy and X-ray diffraction techniques. Examination of silk fibers fragmented with ultrasonic radiation and negatively stained revealed the presence of ribbon-like filaments of well-defined lateral dimensions. Analysis of the breadths of the equatorial reflections in the X-ray diffraction pattern of fibroin yielded similar dimensions for the lateral extent of the crystallites. It is concluded that the crystalline material in B. mori silk fibroin is in the form of ribbon-like filaments of considerable length parallel to the fiber axis and of lateral dimensions approximately 20 x 60 A.     

A Survey of λ Repressor Fragments from Two-State to Downhill Folding

We survey the two-state to downhill folding transition by examining 20 λ_6-85^? mutants that cover a wide range of stabilities and folding rates. We investigated four new λ_6-85^? mutants designed to fold especially rapidly. Two were engineered using the core remodeling of Lim and Sauer, and two were engineered using Ferreiro et al.'s frustratometer. These proteins have probe-dependent melting temperatures as high as 80 °C and exhibit a fast molecular phase with the characteristic temperature dependence of the amplitude expected for downhill folding. The survey reveals a correlation between melting temperature and downhill folding previously observed for the β-sheet protein WW domain. A simple model explains this correlation and predicts the melting temperature at which downhill folding becomes possible. An X-ray crystal structure with a 1.64-Å resolution of a fast-folding mutant fragment shows regions of enhanced rigidity compared to the full wild-type protein.     

Structure and architecture of the bacterial virus fd. an infrared linear dichroism study

Oriented gels of intact bacterial virus fd have been investigated by infrared linear dichroism. Infrared absorption hand maxima and dichroism indicate an α-helix content of the major coat protein of 95–100%. The α-helical rods of the coat protein are aliened parallel to the long axis of the virion with an inclination roughly estimated to ≈37°. The presence of DNA infrared bands at 968, 885. 830 and 799 cm^?, the absence of a hand at 860 cm^?1 and the perpendicular polarization of the symmetric PO₂^? stretching vibration at 1085 cm^?1 are all indicative of a B-type backbone conformation in the single-stranded DNA. We find no evidence for specific interaction between aromatic side groups (phenylalanine. tyrosine) and the DNA bases. Our results independently confirm most features of the model of Marvin and co-workers [2.15] based on low-resolution X-ray diffraction studies. However, our findings contradict their suggestion of an A-type DNA in the bacterial virus fd. Two results are consistent with rigid and stable order in the virus. First, over a 4-day period. 65% of the peptide hydrogens remain unexchanged with deuterium. Second, changes in the relative humidity of the sample do not result in any sliifts in the DNA spectrum that are characteristic of free DNA.     

Stoichiometry and Preferential Interaction: Two Components of the Principle for Protein Structure Organization

Abstract

The effect of pressure on the conformational structure of amyloid β (1–40) peptide (Aβ(1–40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid Aβ(1–40) from 1655 cm^?1 (α-helix) to 1647–1643 cm^?1 (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm^?1 assigned to β-antipar- allel sheet structure was also evident. Furthermore, the peak at 1540 cm^?1 also shifted to 1527 (1529) cm^?1 in amide II band. The former was assigned to the combination of α-helix and random coil structures, and the latter was due to β-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed Aβ(1–40) samples were obtained from 33% to 22% for α-helix/random coil structures and from 47% to 57% for β-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from α-helix to random coil and to β-sheet structure. The structural transformation of the compressed Aβ(1–40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of β-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the β-sheet structure transformed. The thermal-dependent transition temperatures of solid Aβ(1–40) prepared by different pressures were near 55–60 °C.