Changes in the Molecular Packing of Fibrillin Microfibrils During Extension Indicate Intrafibrillar and Interfibrillar Reorganization in Elastic Response Joanna Glab⁎ and Tim Wess School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cardiff CF24 4LU, UK Received 20 June 2008; received in revised form 3 September 2008; accepted 4 September 2008 Available online 12 September 2008 Fibrillin-rich microfibrils are the major structural components of the extracellular matrix that provide elasticity in a majority of connective tissues. The basis of elastic properties lies in the organization of fibrillin molecules, which, unfortunately, is still poorly understood. An X-ray diffraction study of hydrated fibrillin-rich microfibrils from zonular filaments has been conducted to give an insight into the molecular structure of microfibrils in intact tissue. A series of measurements was taken during controlled tissue extension to observe alterations in the lateral packing of microfibrils. Computer-generated simulated patterns were used to fit the experimental X-ray scattering data and to obtain the fibril diameter and lateral distance between the fibrils. The results suggest a nonlinear correlation between external strain and decrease in fibril diameter and lateral spacing. This was accompanied by a nonlinear increase in axial periodicity and a structure with a 160-nm periodicity, which is reported here for the first time using X-ray diffraction. These changes may reflect the unraveling of fibrillin from the complex folded arrangement into a linear structure. This finding supports a pleating model where fibrillin molecules are highly folded within the microfibrils; more importantly, the connection is made between the interaction of individual microfibrils and the change in their suprafibrillar coherent organization during extension. We suggest that the intermediate states observed in our study reflect sequential unfolding of fibrillin and can explain the process of its reversible unraveling. © 2008 Elsevier Ltd. All rights reserved. Edited by M. Moody Keywords: fibrillin; microfibril organization; lateral packing; X-ray diffraction Introduction Fibrillin-rich microfibrils are structural compo- nents of the extracellular matrix that impart elasti- city on a majority of connective tissues. 1,2 Microfibrils are widely distributed in elastin-con- taining tissues such as skin, muscle, lung, ligaments, and vasculature, 3,4 and in nonelastin tissues such as ciliary zonules. 5,6 In elastin-containing tissues, they provide a structural lattice for elastin deposition and are thought to be responsible for normal skin and muscle development, 1,7,8 while in zonules, they are a crucial part of the suspensory system of the lens. 6 Mutations in the fibrillin gene are linked to Marfan syndrome (a severe connective tissue disorder), indicating a key role for microfibrils in the provision of reinforcement and elasticity. 9 Microfibrils have been characterized by electron microscopy (EM) as regular beaded structures with a resting axial periodicity of approximately 56 nm, which is the generally accepted value. 10,11 Micro- fibril diameter has been observed to vary from 10 nm to 40 nm, depending upon the technique used. For instance, an electron microscopic immu- nolocalization study has shown the microfibrils to have an average diameter of 10 nm, 3 whereas rotary shadowing has shown values of 22 nm and 29 nm, 12,13 while atomic force microscopy has yielded a value of 40 nm. 14 It is unclear whether the difference is the result of changes in fibrillin diameter or is the result of the sensitivity of the technique and different methods of microfibril extraction, hydration, and sample preparation. It is *Corresponding author. E-mail address: glabj@cf.ac.uk. Abbreviations used: EM, electron microscopy; SAXS, small-angle X-ray scattering. doi:10.1016/j.jmb.2008.09.004 J. Mol. Biol. (2008) 383, 1171–1180 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.