[Abstract unavailable

The experimental material accumulated in the literature on the conformational behavior of intrinsically unstructured (natively unfolded) proteins was analyzed. Results of this analysis showed that these proteins do not possess uniform structural properties, as expected for members of a single thermodynamic entity. Rather, these proteins may be divided into two structurally different groups: intrinsic coils, and premolten globules. Proteins from the first group have hydrodynamic dimensions typical of random coils in poor solvent and do not possess any (or almost any) ordered secondary structure. Proteins from the second group are essentially more compact, exhibiting some amount of residual secondary structure, although they are still less dense than native or molten globule proteins. An important feature of the intrinsically unstructured proteins is that they undergo disorder-order transition during or prior to their biological function. In this respect, the Protein Quartet model, with function arising from four specific conformations (ordered forms, molten globules, premolten globules, and random coils) and transitions between any two of the states, is discussed.Keywords: Intrinsically unfolded protein; intrinsically disordered protein; unfolded protein; molten globule; premolten globule; partially folded intermediate; random coil; conformational transition This review introduces an intriguing protein family of natively unfolded proteins, whose existence questions one of the cornerstones in protein biology, chemistry and physics, that is, the structure-function paradigm. This concept claims that a specific function of a protein is determined by its unique and rigid three-dimensional (3D) structure. This idea, formulated more than 100 years ago as a lock-and-key model for explaining the amazing specificity of the enzymatic hydrolysis of glucosides (Fischer 1894), proved to be extremely fruitful. Figuratively speaking, the protein structure-function paradigm may be considered as the big bang, creating the universe of modern protein science. Figure 1 attempts to illustrate the most obvious scientific consequences of this concept.However, a reappraisal of the protein structure-function paradigm is now warranted based on systematic studies of intrinsically unfolded/disordered proteins (Wright and Dyson 1999;Dunker et al. 2001;Uversky 2002). There are two major reasons for such a reappraisal: the results of the amino acid sequence analyses and the accumulation of experimental evidence for the existence of a rather large amount of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, both of these sets of evidences were gathered using scientific concepts and approaches originating from the protein structure-function paradigm, namely, proteomics and protein self-organization (see Fig. 1).

Figure 1. Structured domains and intrinsically disordered regions (IDRs) are two fundamental classes of functional building blocks of proteins. The synergy between disordered regions and structured domains increases the functional versatility of proteins. Adapted with permission from ref 50.

"Natively unfolded" proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro. Analysis of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins: small globular folded proteins and "natively unfolded" ones. The results show that "natively unfolded" proteins are specifically localized within a unique region of charge-hydrophobicity phase space and indicate that a combination of low overall hydrophobicity and large net charge represent a unique structural feature of "natively unfolded" proteins.

In the search for the molecular mechanism of insulin fibrillation, the kinetics of insulin fibril formation were studied under different conditions using the fluorescent dye thioflavin T (ThT). The effect of insulin concentration, agitation, pH, ionic strength, anions, seeding, and addition of 1-anilinonaphthalene-8-sulfonic acid (ANS), urea, TMAO, sucrose, and ThT on the kinetics of fibrillation was investigated. The kinetics of the fibrillation process could be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). The addition of seeds eliminated the lag phase. An increase in insulin concentration resulted in shorter lag times and faster growth of fibrils. Shorter lag times and faster growth of fibrils were seen at acidic pH versus neutral pH, whereas an increase in ionic strength resulted in shorter lag times and slower growth of fibrils. There was no clear correlation between the rate of fibril elongation and ionic strength. Agitation during fibril formation attenuated the effects of insulin concentration and ionic strength on both lag times and fibril growth. The addition of ANS increased the lag time and decreased the apparent growth rate for insulin fibril formation. The ANS-induced inhibition appears to reflect the formation of amorphous aggregates. The denaturant, urea, decreased the lag time, whereas the stabilizers, trimethylamine N-oxide dihydrate (TMAO) and sucrose, increased the lag times. The results indicated that both nucleation and fibril growth were controlled by hydrophobic and electrostatic interactions. A kinetic model, involving the association of monomeric partially folded intermediates, whose concentration is stimulated by the air-water interface, leading to formation of the critical nucleus and thence fibrils, is proposed.

Intracellular proteinaceous aggregates (Lewy bodiesand Lewy neurites) of ␣-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying ␣-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that ␣-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant ␣-synuclein. Either a decrease in pH or an increase in temperature transformed ␣-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of ␣-synuclein fibrils. We propose a model for the fibrillation of ␣-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.

No abstract

Intrinsically disordered proteins (IDPs) lack stable tertiary and/or secondary structures under physiological conditions in vitro. They are highly abundant in nature and their functional repertoire complements the functions of ordered proteins. IDPs are involved in regulation, signaling, and control, where binding to multiple partners and high-specificity/low-affinity interactions play a crucial role. Functions of IDPs are tuned via alternative splicing and posttranslational modifications. Intrinsic disorder is a unique structural feature that enables IDPs to participate in both one-to-many and many-to-one signaling. Numerous IDPs are associated with human diseases, including cancer, cardiovascular disease, amyloidoses, neurodegenerative diseases, and diabetes. Overall, intriguing interconnections among intrinsic disorder, cell signaling, and human diseases suggest that protein conformational diseases may result not only from protein misfolding, but also from misidentification, missignaling, and unnatural or nonnative folding. IDPs, such as alpha-synuclein, tau protein, p53, and BRCA1, are attractive targets for drugs modulating protein-protein interactions. From these and other examples, novel strategies for drug discovery based on IDPs have been developed. To summarize work in this area, we are introducing the D2 (disorder in disorders) concept.