SUPRAMOLECULAR STRUCTURES
IN NUCLEOTIDES AND PEPTIDES

 

Understanding the basic ingredients for the formation of ordered structures and mesophases in biological systems is an important challenge for both physicists and biologists.
About this topic, two systems are currently studied in our lab:
- Amyloid fibers formed by poliQ proteins;
- Liquid crystalline phases of concentrated solutions of oligonucletides;

 

Amyloid fibers formed by poliQ proteins

A consequence of marginal stability of proteins is the ease in forming aggregates. In some cases this aggregation is related to the onset of various neurodegenerative diseases, as Alzheimer or Parkinson. In such pathologies some fibrillar aggregates, called amyloids, can be found inside neuronal cells. On the other hand, recent results show that the ability to form ordered fibers is not an unusual property of a few proteins, it’s instead a common characteristic of most of them under proper conditions. It is therefore important to develop general concepts to describe self-association of proteins.

 

TEM images of amorphous and ordered protein aggregates.

 

One of the major challenges for those who study proteins is to describe the mechanisms of cluster formation. We perform Dynamic Light Scattering (DLS) measurements to characterize protein aggregation and we model their kinetic behaviour using concepts from colloidal aggregation.

We study ataxin-3, a protein involved in SCA3, Spinocerebellar Ataxia Type 3, or Machado-Joseph Disease, a neurodegenerative disease. The stability of Ataxin is found to depend on the length of a characteristic poly-glutamine (poly-Q) tract in its sequence. While in the wild-type protein, which is stable in its monomeric form, the poly-Q sequence is shorter than 27 residues, in the pathogenic mutant the repeat is generally longer. We study an expressly modified form of this protein with 36 Q residues, expressed and purified in the laboratory of prof. P. Tortora (University of Milano-Bicocca). In order to explore the self-association mechanism and to look for new therapeutic approaches, we investigate the effect of small molecules, such as Congo Red, on the rate of aggregate formation.

 

Left: 3D structure of Ataxin-3 as proposed by Albrecht et al. (Proteins 50, 355 (2003)); right: Ataxin is composed by a globular domain (Josephin) and a flexible tail containing the poliQ tract.

 

Liquid crystalline phases of concentrated solutions of oligonucletides

The ability of duplex DNA to form liquid crystal (LC) phases when hydrated has been known since the late 1940’s and played a crucial role in the decipherment of its structure, enabling measurement of the x-ray structure factor of single B-DNA chains uncomplicated by inter-chain correlations. Since that time the LC phases of solutions of duplex DNA have been extensively characterized by optical, x-ray and magnetic resonance methods for chain lengths, N, ranging from mega base pair (bp) semiflexible polymers down to nucleosomal N ~ 150 bp rigid rod-like segments. These studies have revealed, at temperatures T below the duplex melting and vs. increasing DNA concentration:  isotropic (I); chiral nematic (N*); uniaxial columnar (CU); higher-ordered columnar (C1); and crystal (X) phases of B-DNA.

 

Crystalline (left) and liquid-crystalline (right) phases in DNA.

We can observe LC phases also in very short fragments of DNA: cholesteric (a) and columnar (b,c); at higher concentrations an amorphous solid (glassy?) state appears.

 

The appearance of such LC phases in DNA solutions has been accounted for theoretically by modelling the DNA as a repulsive rod-shaped or semiflexible polymer solute which, at sufficiently high volume fraction f, experiences a net entropy gain upon orientational ordering in the trade-off between orientational and translational entropy. The basic model is Onsager's treatment of the formation of the nematic phase in a system of monodisperse repulsive hard rods (length L, diameter D), in which nematic ordering appears for volume fraction f > f_IN = 4D/L. Below this value LC phases should never be expected in actual DNA solutions, since the generalizations of Onsager's model, believed to be required to describe DNA, such as introduction of chain flexibility, electrostatic or hydration repulsion, or polydispersity, all have the effect of suppressing nematic order relative to isotropic.

It was therefore quite surprising for us to find a various mesophases in B-DNA duplexes of very short length, an order of magnitude smaller than expected from f_IN. In seeking an explanation of these observations we are currently characterizing LC phases in oligonucleotides using depolarized transmission light microscopy, optical interferometry, X-ray diffraction and fluorescence measurements. This project is carried out in collaboration with Prof. Noel Clark of the Condensed Matter Laboratory in Boulder (CO).

 

Phase transition occurring from cholesteric to columnar phase in oligonucleotide.