Titin; a multidomain protein that behaves as the sum of its parts¹

Abstract

Titin is a giant, multidomain muscle protein forming a major component of the sarcomere in vertebrate striated muscle. As for many other multidomain proteins, the properties of titin are often studied by characterisation of the constituent domains in isolation. This raises the question of to what extent the properties of the isolated domains are representative of the domains in the wild-type protein. We address this question for the I-band region of titin, which is of particular biological interest due to its role in muscle elasticity, by determining the properties of five immunoglobulin domains from the I-band in three different contexts; firstly as isolated domains with the boundaries defined conservatively, secondly, with a two amino acid extension at both the N and C terminus and thirdly as part of multidomain constructs. We show that adjacent domains in the titin I-band have very different kinetic properties which, in general, undergo only a small change in the presence of neighbouring domains and conclude that, provided that care is taken in the choice of domain boundaries, the properties of the titin I-band are essentially “the sum of its parts”. From this and other work we propose that variation in kinetic properties between adjacent domains may be a general property of the I-band thereby preventing misfolding events on muscle relaxation.

Introduction

Advances in structural genomics indicate that a significant fraction of the proteins encoded by genomes contain two or more domains.1 For small proteins with a limited number of domains it is sometimes possible for the properties of the domains to be determined directly in the context of the wild-type protein, however, for large, multidomain proteins this is often not possible using current methods. As a result they are typically studied by characterisation of the constituent domains in isolation. This raises questions as to what extent the properties of the isolated domains are representative of the domains in the wild-type protein and how domain-domain interactions modulate the properties of single domains (see for example2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15).

One such multidomain protein that has been studied by characterisation of its domains in isolation is titin. Titin is a giant, 3 MDa, protein and a major constituent of the sarcomere in vertebrate striated and cardiac muscle.16, 17 In addition to forming a scaffold for the assembly of muscle thick filaments titin plays an important role in muscle elasticity and the generation of passive tension.16, 18, 19 Titin is divided into two regions; the A-band and the I-band. Thick filament assembly is attributed to the A-band region of titin which interacts with both the M-line proteins and the thick filaments acting as a template for assembly. The I-band, which acts as an elastic connector between the A-band and the Z disk, is believed to be responsible for the elastic properties of titin20, 21, 22 and thus is the region of interest in experiments to determine the basis of muscle elasticity. The I-band consists of a tandem array of immunoglobulin (Ig) domains interrupted by less structured linker sequences;23 the largest of these linker sequences is the PEVK region, rich in the amino acids proline, glutamic acid, valine and lysine. At low forces the elastic properties of titin are consistent with the Ig domains and the PEVK region acting as an entropic spring,18, 20, 23, 24 whilst at higher forces elasticity is attributed to the un-folding of the Ig domains (references include25, 26, 27, 28, 29); the number of Ig domains which unfold has recently been estimated using simulation.30 It has been shown, for titin, that several isolated Ig domains and Ig domains expressed in tandem are fully folded in solution and that Ig domains expressed in tandem unfold independently.31, 32, 33, 34, 35 However, questions remain as to the extent of interactions between domains and the effect that any such interactions may have on the properties of the domains.

To what extent are the isolated Ig domains a good model for the properties and behaviour of the domains in titin? Previous studies have shown that when Ig domains are expressed in tandem both domains show an increase in stability, with the increase being most significant for the less stable of the two domains.33, 34, 36 This increase in stability has been attributed to the effect of domain-domain interactions.33, 34 However, comparison of data from experiments using chemical denaturation and atomic force microscopy suggests that the increase in stability may not be entirely due to domain-domain interactions. The 27th and 28th domains of human cardiac titin (I27 and I28) have been studied in detail using both chemical denaturation and atomic force microscopy (AFM).34, 37 Chemical denaturation studies show that the stability of I28 is increased when expressed in tandem with I27. In addition the rate of unfolding of I28 measured by both AFM and chemical denaturation is decreased in I2728 constructs. In the AFM experiment all of the I27 domains in the (I2728)₄ construct unfold before the first I28 domain, whilst in chemical denaturation experiments at low concentrations of denaturant I27 remains folded under conditions where I28 unfolds. This shows that the presence of I27 modulates the properties of I28 whether I27 is folded or not. It is therefore possible that the observed changes in the properties of I28 are as a result of the presence of additional residues at the N terminus rather than domain-domain interactions. (Stabilisation by extension at the N and C terminus is not a novel observation; it has been shown that the stability of a number of isolated domains, including Ig and FnIII domains, increases upon the extension of the N terminus.36, 38, 39)

Here we build on previous work characterising the interactions between tandem titin Ig domains19, 22 by measuring the contribution of the additional residues at the N and C termini to the properties of isolated titin domains. We then compare the relative effects of the inclusion of the additional residues and of domain-domain interactions on the properties of domains in tandem constructs. To do this we determine the properties of five titin Ig domains in three different contexts. Firstly, the domains are expressed with boundaries defined using the multiple sequence alignment by Pastore and co-workers,21 secondly, with a two amino acid extension at both the N and C terminus, ensuring that linker residues are present at both termini, and thirdly as part of multidomain constructs.