Elastic fibres are an important component of the extracellular matrix and are made of two major components : the more abundant cross-linked elastic protein elastin and the multi-component microfibrils. The biosynthesis of elastic fibres is a complex process involving the interplay of many diverse proteins and genes with elastin as the major component. Tropoelastin is the soluble precursor of elastin and as such it plays a dominant role in elastogenesis. The expression of tropoelastin is under a complex control mechanism, with many isoforms existing. Numerous other components, including the microfibrillar proteins, the elastin-binding protein and lysyl oxidase, the enzyme which initiates elastin cross-linking, are involved in elastogenesis. Tropoelastin undergoes self-association under physiological conditions in a process referred to as coacervation, and this is thought to be a vital process during elastic fibre formation and in providing elasticity. Although various models explaining the elasticity of elastin have been put forward, only the fibrillar model is based on the coacervation ability of tropoelastin. With the molecular cloning of a number of components of the elastic fibre, the availability of these components is increasing and paves the way for in vitro modelling of complex interactions of the elastic fibre. This review emphasises the biochemistry of tropoelastin and its role in elastic fibre structure and assembly.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.1998.2580001.x

Biochemistry of tropoelastin

The intent of this paper is to bring up to date an earlier theory of equilibrium selectivity [23, 24], with emphasis on making it applicable to the permeation of membrane channels and carriers, which involve kinetic considerations [8, 56]. We will review critically and unify a number of conceptual advances that have occurred since 1961, particularly the recognition by Hille [56] of how the same energetic principles that describe equilibrium selectivity of binding sites also apply to the peaks of the energy barriers (the so-called selectivity filters) involved in the kinetics of permeation. In the process, the precision of the term selectivity will gradually be increased. We will start with the intuitively simple comparison of effects of one ion versus another. Then we will proceed through more theoretically based classical concepts such as permeability ratios, conductance ratios, and ratios of binding affinities, all of which can be interrelated quite directly in sufficiently simple channels. It will become clear that these concepts lose their crispness and usefulness in multi-barrier channels as a consequence of asymmetry and/or possible multiple occupancy. Our formulation is influenced by a number of useful concepts from classical rate theory [40, 48, 126, 128], where the channel is viewed as having a static energy profile for a given state of ion occupancy; but we will also consider the consequences of viewing the channel as a dynamic structure with a fluctuating energy profile [83, 851. Besides presenting a topical review of selectivity, we have found it necessary to consider two subjects which are so new that they have received little attention as yet in a selectivity context. These are the effects in multi-barrier channels of asymmetry and multiple occupancy. This is because a number of classical concepts, and even ways of thinking about selectivity, are implicitly conditioned by conclusions that have been reached from considerations heretofore largely restricted to channels occupiable by no more than one ion at a time, or to channels with only one significantly rate determining barrier 1, or to channels which are symmetrical. To exemplify the effects of channel asymmetry, we examine in a simple two-barrier one-site model the consequences of asymmetry for reversal potential selectivity. To exemplify the effects of multiple rate-determining barriers, we also examine some implications of the energy profile which can be inferred for the gramicidin channel. In the process of presenting this material, we describe certain relationships between selectivity as seen in different phenomena such as conductance, reversal potential, and binding at least in systems which are simple enough for these classical concepts to retain their utility. This should lead to an understanding of why different measures of selectivity sometimes lead to different apparent selectivities, even in very simple channels. We also will define some guidelines for the applicability of these classical concepts and give some suggestions as to how to define selectivity when they fail, as fail they must, in those biological channels which are asymmetric and/or occupied by more than one ion at a time.

Ionic selectivity revisited: The role of kinetic and equilibrium processes in ion permeation through channels

We have discussed in some detail a variety of experimental studies which were designed to elucidate the conformational and dynamic properties of gramicidin and alamethicin. Although the behavior of these peptides is by no means fully characterized, these studies have already permitted aspects of ion channel activity to be understood in molecular terms. Studies with gramicidin in a variety of organic solutions have revealed conformational heterogeneity of this peptide; at least five major isomers exist, several of which have been characterized in detail using NMR spectroscopy and X-ray crystallography. When added to lipid membranes gramicidin undergoes a further conformational conversion. Although the conformation of gramicidin in membranes is not as well characterized as the solution conformation(s) and an X-ray structure is not yet available, detailed data, particularly from solid-state NMR studies, continue to become available and a right-handed beta 6.3 helical conformation of the peptide backbone is now generally accepted. Two of these beta 6.3 helices joined at their N-termini are believed to form the conducting channel. The conformational behavior of the side-chains of gramicidin in the membrane-bound form is not well established and several NMR, CD, fluorescence and theoretical studies are now focussed on this. Although the side-chains do not directly contact the permeating ions, they can have distinct effects on conductance and selectivity by altering the electrostatic environment sensed by the ion. The dynamics of both side-chain and backbone conformations of gramicidin appear critical to a detailed understanding of the ion transport process in this channel. As the description of the membrane-bound conformation of gramicidin becomes more detailed, simulations of ion transport using computational methods are likely to improve and will further our understanding of the processes of ion transport. As well as internal motion of the backbone and side-chains, gramicidin undergoes rotational and translational motion in the plane of the membrane. These motions do not appear to be essential for the process of ion transport but can affect channel lifetime since lifetime is determined by the rate of association and dissociation of gramicidin monomers. Gramicidin-membrane interactions are also likely to be involved in the frequency of occurrence of channel subconductance states, the frequency of channel flickering and fundamentally in the stability of the membrane-bound gramicidin conformation. Alamethicin forms channels in membranes which are strongly voltage-dependent. The molecular origin of voltage-dependent conductances has been a fundamental problem in biophysics for many years.(ABSTRACT TRUNCATED AT 400 WORDS)

Model ion channels: gramicidin and alamethicin.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2885%2980702-X

1H-NMR study of gramicidin A transmembrane ion channel: Head-to-head right-handed, single-stranded helices

In general, proteins fold with hydrophobic residues buried, away from water. Reversible protein folding due to hydrophobic interactions results from inverse temperature transitions where folding occurs on raising the temperature. Because homoiothermic animals constitute an infinite heat reservoir, it is the transition temperature, Tt, not the endothermic heat of the transition, that determines the hydrophobically folded state of polypeptides at body temperature. Reported here is a new hydrophobicity scale based on the values of Tt for each amino acid residue as a guest in a natural repeating peptide sequence, the high polymers of which exhibit reversible inverse temperature transitions. Significantly, a number of ways have been demonstrated for changing Tt such that reversibly lowering Tt from above to below physiological temperature becomes a means of isothermally and reversibly driving hydrophobic folding. Accordingly, controlling Tt becomes a mechanism whereby proteins can be induced to carry out isothermal free energy transduction.

Hydrophobicity scale for proteins based on inverse temperature transitions

Thallium ion-induced carbonyl carbon chemical shifts were compared for all of the L-residue-peptide carbonyl carbons of the gramicidin A transmembrane channel. Molecular structures were deduced by using the argument that helically equivalent and equally proximal carbonyls would exhibit essentially equivalent ion-induced chemical shifts. The transmembrane channel was found to be a head-to-head dimer with the structure of a left-handed, single-stranded β-helix.

https://science.sciencemag.org/content/sci/221/4615/1064.full.pdf

Is the Gramicidin A Transmembrane Channel Single-Stranded or Double-Stranded Helix? A Simple Unequivocal Determination

Malonyl gramicidin is incorporated into lysolecithin micelles in a manner which satisfies a number of previously demonstrated criteria for the formation of the transmembrane channel structure. By means of sodium-23 nuclear magnetic resonance, two binding sites are observed: a tight site and a weak site with binding constants of approximately 100m
−1 and 1m
−1, respectively. In addition, off-rate constants from the two sites were estimated from NMR analyses to bek

 off
 ≃3×105/sec andk

 off
 ≃2×107/sec giving, with the binding constants, the on-rate constants,k

 on
 ≃3×107/msec andk

 on
 ≃2×107/m sec. Five different multiple occupancy models with NMR-restricted energy profiles were considered for the purpose of calculating single-channel currents as a function of voltage and concentration utilizing the four NMR-derived rate constants (and an NMR-limit placed on a fifth rate constant for intrachannel ion translocation) in combination with Eyring rate theory for the introduction of voltage dependence. Using the X-ray diffraction results of Koeppe et al. (1979) for limiting the positions of the tight sites, the two-site model and a three-site model in which the weak sites occur after the tight site is filled were found to satisfactorily calculate the experimental currents (also reported here) and to fit the experimental currents extraordinarily well when the experimentally derived values were allowed to vary to a least squares best fit. Surprisingly the “best fit” values differed by only about a factor of two from the NMR-derived values, a variation that is well within the estimated experimental error of the rate constants. These results demonstrate the utility of ion nuclear magnetic resonance to determine rate constants relevant to transport through the gramicidin channel and of the Eyring rate theory to introduce voltage dependence.

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

Prolyl hydroxylation of the polypentapeptide model of elastin impairs fiber formation.

By means of carbon-13 nuclear magnetic resonance, carbonyl carbon ion-induced chemical shifts of the Gramicidin A channel are determined at 70 °C and analyzed as a function of potassium ion concentration and as a function of thallium ion concentration. In both cases two binding processes are observed. The estimated binding constants for potassium ion binding are 28/M and 2.4/M for the tight (single ion occupancy) and weak (double ion occupancy) sites, respectively, and for thallium ion binding are of the order of 103/M for the tight site and approximately 70/M for the weak site. These studies, which utilize a 90% enriched carbonyl carbon within the binding site of the channel to monitor ion interaction, argue for the presence of two ions in the channel for conditions under which planar bilayer transport studies are commonly carried out.

K. U. Prasad

Papers

Is the Gramicidin A Transmembrane Channel Single-Stranded or Double-Stranded Helix? A Simple Unequivocal Determination

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

Prolyl hydroxylation of the polypentapeptide model of elastin impairs fiber formation.

Carbon-13 nuclear magnetic resonance study of potassium and thallium ion binding to the Gramicidin A transmembrane channel

The source of the dispersity of gramicidin A single-channel conductances. The L X Leu5-gramicidin A analog