Tài liệu Báo cáo Y học: A Raman optical activity study of rheomorphism in caseins, synucleins and

A Raman optical activity study of rheomorphism in caseins,

synucleins and tau

New insight into the structure and behaviour of natively unfolded proteins

Christopher D. Syme1

, Ewan W. Blanch1

, Carl Holt2

, Ross Jakes3

, Michel Goedert3

, Lutz Hecht1

and Laurence D. Barron1

1

Department of Chemistry, University of Glasgow, UK; 2

Hannah Research Institute, Ayr, UK; 3

Medical Research Council Laboratory

of Molecular Biology, Cambridge, UK

The casein milk proteins and the brain proteins a-synuclein

and tau have been described as natively unfolded with ran￾dom coil structures, which, in the case of a-synuclein and tau,

have a propensity to form the ®brils found in a number of

neurodegenerative diseases. New insight into the structures

of these proteins has been provided by a Raman optical

activity study, supplemented with di€erential scanning cal￾orimetry, of bovine b- and j-casein, recombinant human a-,

b- and c-synuclein, together with the A30P and A53T mu￾tants of a-synuclein associated with familial cases of Par￾kinson's disease, and recombinant human tau46 together

with the tau46 P301L mutant associated with inherited

frontotemporal dementia. The Raman optical activity

spectra of all these proteins are very similar, being dominated

by a strong positive band centred at » 1318 cm)1 that may be

due to the poly(L-proline) II (PPII) helical conformation.

There are no Raman optical activity bands characteristic of

extended secondary structure, although some unassociated

b strand may be present. Di€erential scanning calorimetry

revealed no thermal transitions for these proteins in the

range 15±110 °C, suggesting that the structures are loose and

noncooperative. As it is extended, ¯exible, lacks intrachain

hydrogen bonds and is hydrated in aqueous solution, PPII

helix may impart a rheomorphic (¯owing shape) character to

the structure of these proteins that could be essential for their

native function but which may, in the case of a-synuclein and

tau, result in a propensity for pathological ®bril formation

due to particular residue properties.

Keywords: caseins, synucleins and tau; polyproline II helix;

amyloid ®brils; neurodegenerative disease; Raman optical

activity.

Although nonregular protein structures are usually encoun￾tered under certain denaturing conditions, it is becoming

increasingly apparent that proteins with nonregular struc￾tures also exist under physiological conditions [1]. The fact

that such proteins can have important biological functions

has necessitated a reassessment of the structure±function

paradigm [2]. Native proteins with nonregular structures

include the casein milk proteins [3], the phosphophoryns of

bone and the phosvitins of egg yolk [4], Bowman±Birk

protease inhibitors [5], metallothioneins [6], prothymosin

a [7], a bacterial ®bronectin-binding protein [8], the brain

protein a-synuclein together with the related proteins

b-synuclein and c-synuclein [9±12], and the brain protein

tau [13±16]. In addition to their role in normal function,

nonregular protein structures in both non-native and native

states are also of interest on account of their susceptibility to

the type of aggregation found in many protein misfolding

diseases.

The heterogeneity of nonregular protein structures, non￾native or native, has made their detailed characterization

dif®cult. As a result, all nonregular protein structures are

often called random coil, implying that they behave like

synthetic high polymers in dilute aqueous solution for which

the random coil model was originally developed. The

random coil state is envisaged as the collection of an

enormous number of possible random conformations of an

extremely long molecule in which chain ¯exibility arises

from internal rotation (with some degree of hindrance)

around the covalent backbone bonds [17]. However, there is

a growing awareness that this extreme situation does not

occur in most nonregular protein states. In order to further

our understanding of the behaviour of proteins with

nonregular structures, it is necessary to employ experimental

techniques able to discriminate between the dynamic true

random coil state and more static types of disorder.

One such technique is Raman optical activity (ROA),

which measures vibrational optical activity by means of a

small difference in the intensity of Raman scattering from

chiral molecules in right- and left-circularly polarized

incident laser light [18]. It has recently been demonstrated

that ROA is able to distinguish two distinct types of

disorder in nonregular protein structures in aqueous solu￾tion [19]. The delimiting cases are a dynamic disorder

corresponding to that envisaged for the random coil in

Correspondence toL. D. Barron, Department of Chemistry, University

of Glasgow, Glasgow G12 8QQ, UK. Fax: + 44 141 330 4888,

Tel.: + 44 141 330 5168, E-mail: [email protected]

Abbreviations: DSC, di€erential scanning calorimetry; PPII, poly(L￾proline) II; ROA, Raman optical activity; UVCD, ultraviolet circular

dichroism; VCD, vibrational circular dichroism.

Note: a web site is available at http://www.chem.gla.ac.uk

(Received 5 September 2001, revised 18 October 2001, accepted 25

October 2001)

Eur. J. Biochem. 269, 148±156 (2002) Ó FEBS 2002