The intermediate filament architecture as determined by X-ray diffraction modeling of hard alpha-keratin (original) (raw)
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The Intermediate Filament Architecture as Determined by X-Ray Diffraction Modeling of Hard α-Keratin
Biophysical Journal, 2004
Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard a-keratin intermediate filaments under in vivo conditions. In short, the a-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.
Journal of Structural Biology, 2006
Several aspects of the intermediate filaments' molecular architecture remain mysterious despite decades of study. The growth process and the final architecture may depend on the physical, chemical, and biochemical environment. Aiming at clarifying this issue, we have revisited the structure of the human hair follicle by means of X-ray microdiffraction. We conclude that the histology-based growth zones along the follicle are correlated to the fine architecture of the filaments deduced from X-ray microdiffraction. Our analysis reveals the existence of two major polymorph intermediate filament architectures. Just above the bulb, the filaments are characterized by a diameter of 100 Å and a low-density core. The following zone upwards is characterized by the lateral aggregation of the filaments into a compact network of filaments, by a contraction of their diameter (to 75 Å ) and by the setting up of a long-range longitudinal ordering. In the upper zone, the small structural change associated with the tissue hardening likely concerns the terminal domains. The architecture of the intermediate filament in the upper zones could be specific to hard a-keratin whilst the other architecture found in the lower zone could be representative for intermediate filaments in a different environment.
Biochemistry Research International, 2011
High-and low-angle X-ray diffraction studies of hard α-keratin have been studied, and various models have been proposed over the last 70 years. Most of these studies have been confined to one or two forms of alpha keratin. This high-and low-angle synchrotron fibre diffraction study extends the study to cover all available data for all known forms of hard α-keratin including hairs, fingernails, hooves, horn, and quills from mammals, marsupials, and a monotreme, and it confirms that the model proposed is universally acceptable for all mammals. A complete Bragg analysis of the meridional diffraction patterns, including multiple-time exposures to verify any weak reflections, verified the existence of a superlattice consisting of two infinite lattices and three finite lattices. An analysis of the equatorial patterns establishes the radii of the oligomeric levels of dimers, tetramers, and intermediate filaments (IFs) together with the centre to centre distance for the IFs, thus confirming the proposed helices within helices molecular architecture for hard α-keratin. The results verify that the structure proposed by Feughelman and James meets the criteria for a valid α-keratin structure.
The intermediate filament structure of human hair
Biochimica et Biophysica Acta (BBA) - General Subjects, 1995
X-ray diffraction studies of hard a-keratin have led to a proposed model lbr the lateral arrangement of molecules within the keratin fibrils of tissues such as hair, nail and claw. Using low-angle synchrotron radiation to examine human scalp hair we have obtained discrete equatorial diffraction maxima which have not been reported previously. These reflections can be divided into three subsets. The first of these reveals the information that the hair fibres consist basically of cylindrical fibrils arranged in a disordered lattice. The mean diameters of these cylinders have been determined, together with their average separation. The diameters of the protofibrils have been determined from the second set. The third set, a set of diffuse arcs, index onto a spacing which is characteristic of the disordered components of the matrix.
Organization of microfibrils in keratin fibers studied by X-ray scattering
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1998
Low-angle X-ray scattering patterns of hard alpha-keratin fibers have been studied for more than 50 years but a completely convincing modelling has never been presented. The models which have been proposed so far are specific to the sample and cannot be adapted to others, mainly because they do not use a parametric analytical expression of the distribution function describing the relative positions of the microfibrils. Our new approach is based on a paracrystal distribution function. In addition, a huge background originating from a non-ordered matrix is taken into account. Various hard alpha-keratins from different origins have been studied using our approach. From the rather good modellings obtained, it appears that the diameter of the microfibril is not origin dependent (7.4 nm) whereas the distances between microfibrils and their electron density profiles are. Hair microfibrils can be reasonably approximated by a solid cylinder but a core and an outer ring are necessary for porcupine. Our method is of course not limited to keratin microfibrils; it can be used for modelling equatorial X-ray scattering profiles of all types of hexagonal fibrillar assemblies, which are in fact widely found in biological tissues. ß 1998 Elsevier Science B.V. All rights reserved.
The Journal of Cell Biology, 1983
We have used scanning transmission electron microscopy to elucidate the question of how intermediate filament (IF) subunits of widely differing mass can all form morphologically similar IF. From scanning transmission electron micrographs, the distributions of mass were determined for three types of epidermal keratin IF reassembled in vitro from mixtures of subunits with substantially different masses, viz., "light" and "heavy" human keratins with [Mr] = 50,000 and 56,000, respectively, and mouse keratins of [Mr] = 63,000. Their principal assembly products were found to average 22, 25, and 29 kdalton/nm, respectively. These densities, which correspond to immature "minimal form" IF (Steven, A. C., J. Wall, J. Hainfeld, and P. M. Steinert, 1982, Proc. Natl. Acad. Sci. USA., 79:3101-3105), are directly proportional to the average subunit masses. The human keratin IF (but not those of mouse) also contained minor amounts (15-20%) of more massive polymers aver...
Elucidating the early stages of keratin filament assembly
The Journal of Cell Biology, 1990
Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed human K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel. 1. Abbreviations used in this paper: 1B, inclusion body; IBF, IB-rich fraction; IE intermediate filament. formed with pETK14; (lane 10) IBF from bacterial extract in lane 9; (lane 11) Mono Q purification of K14 IBF in lane 10; and (lane 12) Superose 12 purification of pooled Mono Q fractions from lane 11. Molecular masses of epidermal keratins are indicated at left.
Biophysical Journal, 2002
The mechanical behavior of human hair fibers is determined by the interactions between keratin proteins structured into microfibrils (hard ␣-keratin intermediate filaments), a protein sulfur-rich matrix (intermediate filaments associated proteins), and water molecules. The structure of the microfibril-matrix assembly has already been fully characterized using electron microscopy and small-angle x-ray scattering on unstressed fibers. However, these results give only a static image of this assembly. To observe and characterize the deformation of the microfibrils and of the matrix, we have carried out time-resolved small-angle x-ray microdiffraction experiments on human hair fibers stretched at 45% relative humidity and in water. Three structural parameters were monitored and quantified: the 6.7-nm meridian arc, which is related to an axial separation between groups of molecules along the microfibrils, the microfibril's radius, and the packing distance between microfibrils. Using a surface lattice model of the microfibril, we have described its deformation as a combination of a sliding process and a molecular stretching process. The radial contraction of the matrix is also emphasized, reinforcing the hydrophilic gel nature hypothesis.
New Aspects of the α-Helix to β-Sheet Transition in Stretched Hard α-Keratin Fibers
Biophysical Journal, 2004
The putative transformation of a-helices into b-sheets has been studied for more than 50 years in the case of hard a-keratin. In a previous study of stretched keratin fibers, we specified the conditions for b-sheet appearance within horsehair: the formation of b-sheets requires at least 30% relative humidity. However, this phenomenon was observed in the whole tissue. Then there was no clear chemical identification of the b-sheets (keratin or matrix proteins) and the exact location of the b-sheets across the fiber could not be specified. In this study, using wide-angle x-ray scattering and high spatial resolution infrared microspectroscopy, we could determine and characterize the structural elements across hair sections stretched in water, which provides new information about the aforementioned transition. Our results show that the process can be split into three steps: 1), unraveling of the a-helical coiled-coil domains, which starts at roughly 5% macroscopic strain; 2), further transformation of the unraveled coiled-coils into b-sheet structures, which occurs above roughly 20% macroscopic strain; and 3), spatial expanding of the b-structured zones from the sample center to its periphery.
Biochimica et Biophysica Acta (BBA)-Protein …, 1998
Low-angle X-ray scattering patterns of hard alpha-keratin fibers have been studied for more than 50 years but a completely convincing modelling has never been presented. The models which have been proposed so far are specific to the sample and cannot be adapted to others, mainly because they do not use a parametric analytical expression of the distribution function describing the relative positions of the microfibrils. Our new approach is based on a paracrystal distribution function. In addition, a huge background originating from a non-ordered matrix is taken into account. Various hard alpha-keratins from different origins have been studied using our approach. From the rather good modellings obtained, it appears that the diameter of the microfibril is not origin dependent (7.4 nm) whereas the distances between microfibrils and their electron density profiles are. Hair microfibrils can be reasonably approximated by a solid cylinder but a core and an outer ring are necessary for porcupine. Our method is of course not limited to keratin microfibrils; it can be used for modelling equatorial X-ray scattering profiles of all types of hexagonal fibrillar assemblies, which are in fact widely found in biological tissues. ß 1998 Elsevier Science B.V. All rights reserved.