Collagen is probably the most considerable protein in animals. This fibrous, structural protein includes a right-handed bundle of three parallel, left-handed polyproline II-type helices. A lot progress has been made in elucidating the construction of collagen triple helices and the physicochemical foundation for his or her stability. New proof demonstrates that stereoelectronic results and preorganization play a key function in that stability. The fibrillar construction of sort I collagen–the prototypical collagen fibril–has been revealed intimately. Synthetic collagen fibrils that show some properties of pure collagen fibrils are actually accessible utilizing chemical synthesis and self-assembly. A quickly rising understanding of the mechanical and structural properties of native collagen fibrils will information additional growth of synthetic collagenous supplies for biomedicine and nanotechnology.
The classes of collagen embody the classical fibrillar and network-forming collagens, the FACITs (fibril-associated collagens with interrupted triple helices), MACITs (membrane-associated collagens with interrupted triple helices), and MULTIPLEXINs (a number of triple-helix domains and interruptions). Collagen sorts, their distribution, composition, and pathology are listed in . It’s noteworthy that, though the three polypeptide chains within the triple helix of every collagen sort might be equivalent, heterotrimeric triple helices are extra prevalent than are homotrimeric triple helices.
The defining function of collagen is a chic structural motif through which three parallel polypeptide strands in a left-handed, polyproline II-type (PPII) helical conformation coil about one another with a one-residue stagger to kind a right-handed triple helix ( ). The tight packing of PPII helices throughout the triple helix mandates that each third residue be Gly, leading to a repeating XaaYaaGly sequence, the place Xaa and Yaa might be any amino acid. This repeat happens in all forms of collagen, though it’s disrupted at sure places throughout the triple-helical area of nonfibrillar collagens ( 8 ). The amino acids within the Xaa and Yaa positions of collagen are sometimes (2S)-proline (Professional, 28%) and (2S,4R)-4-hydroxyproline (Hyp, 38%), respectively. ProHypGly is the most typical triplet (10.5%) in collagen ( 9 ). In animals, particular person collagen triple helices, referred to as tropocollagen (TC), assemble in a fancy, hierarchical method that finally results in the macroscopic fibers and networks noticed in tissue, bone, and basement membranes ( ).
Collagen is an considerable structural protein in all animals. In people, collagen includes one-third of the overall protein, accounts for three-quarters of the dry weight of pores and skin, and is probably the most prevalent part of the extracellular matrix (ECM). Twenty-eight various kinds of collagen composed of at the very least 46 distinct polypeptide chains have been recognized in vertebrates, and lots of different proteins comprise collagenous domains ( 1 , 2 ). Remarkably, intact collagen has been found in delicate tissue of the fossilized bones of a 68 million-year-old Tyrannosaurus rex fossil ( 3 , 4 ), by far the oldest protein detected up to now. That discovery is, nonetheless, underneath problem ( 5 , 6 ).
Most X-ray crystallographic research on CRPs have been carried out on proline-rich collagenous sequences. The entire ensuing constructions have a 7/2 helical pitch (20.0-Å axial repeat), in distinction to the ten/3 helical pitch (28.6-Å axial repeat) predicted for pure collagen by fiber diffraction ( 17 ). On the idea of X-ray crystal constructions of proline-rich CRPs, and in accordance with an early proposal relating to the helical pitch of pure triple helices ( 23 ), Okuyama and coworkers ( 24 ) postulated that the proper common helical pitch for pure collagen is 7/2. The generality of this speculation is unclear, as few areas of pure collagen are as proline wealthy because the CRPs analyzed by X-ray crystallography. The precise helical pitch of collagen possible varies throughout the domains and forms of pure collagen. Particularly, the helical pitch may very well be 10/3 in proline-poor areas and seven/2 in proline-rich areas. This proposal is supported by the statement that proline-poor areas inside crystalline CRPs often show a ten/3 helical pitch ( 25 , 26 ). Variability within the triple-helical pitch of native collagen may play a job within the interplay of collagenous domains with different biomolecules ( 22 , 27 – 29 ).
In 1994, Berman and coworkers ( 19 ) reported the primary high-resolution crystal construction of a triple-helical CRP ( ). This construction confirmed the existence of interstrand N– H (Gly) ⋯O=C (Xaa) hydrogen bonds ( ) and supplied further insights, together with that C α –H (Gly/Yaa) ⋯O=C (Xaa/Gly) hydrogen bonds may likewise stabilize the triple helix ( 20 ). Utilizing CRPs and X-ray crystallography, the structural affect of a single Gly → Ala substitution was noticed ( 19 ), the results of neighboring charged residues in a triple helix had been analyzed ( 21 ), and a snapshot of the interplay of a triple-helical CRP with the I– area of integrin α 2 β 1 was obtained ( ) ( 22 ).
Fiber diffraction research can not reveal the construction of collagen at atomic decision. Exacerbating this problem, the big dimension, insolubility, repetitive sequence, and complicated hierarchical construction of native collagen thwart most biochemical and biophysical analyses. Therefore, a reductionist strategy utilizing triple-helical, collagen-related peptides (CRPs) has been employed extensively because the late Sixties ( 18 ).
In 1940, Astbury & Bell ( 11 ) proposed that the collagen molecule consists of a single prolonged polypeptide chain with all amide bonds within the cis conformation. A big advance was achieved when, in the identical 1951 subject of the Proceedings of the Nationwide Academy of Sciences through which he and coworkers put forth the proper constructions for the α-helix and β-sheet, Pauling & Corey ( 12 ) proposed a construction for collagen. In that construction, three polypeptide strands had been held collectively in a helical conformation by hydrogen bonds. Inside every amino acid triplet, these hydrogen bonds engaged 4 of the six primary chain heteroatoms, and their formation required two of the three peptide bonds to be within the cis conformation. In 1954, Ramachandran & Kartha ( 13 , 14 ) superior a construction for the collagen triple helix on the idea of fiber diffraction knowledge. Their construction was a right-handed triple helix of three staggered, left-handed PPII helices with all peptide bonds within the trans conformation and two hydrogen bonds inside every triplet. In 1955, this construction was refined by Wealthy & Crick ( 15 – 16 ) and by North and coworkers ( 17 ) to the triple-helical construction accepted right this moment–, which has a single interstrand N–H (Gly) ⋯O=C (Xaa) hydrogen bond per triplet and a tenfold helical symmetry with a 28.6-Å axial repeat (10/3 helical pitch) ( ).
UNDERSTANDING TRIPLE-HELIX STRUCTURE AND STABILITY
The important significance of collagen as a scaffold for animals calls for a manifold of important traits. These traits embody thermal stability, mechanical power, and the flexibility to interact in particular interactions with different biomolecules. Understanding how such properties are derived from the elemental structural unit of collagen, the triple helix, necessitates a complete information of the mechanisms underlying triple-helix construction and stability.
Interstrand Hydrogen Bonds The ubiquity of collagen makes the ladder of recurrent N–H (Gly) ⋯O=C (Xaa) hydrogen bonds that kind throughout the triple helix ( ) probably the most considerable amide–amide hydrogen bond in kingdom Animalia. Changing the Yaa–Gly amide bond with an ester in a host-guest CRP ( ) enabled estimation of the power of every amide–amide hydrogen bond as ΔG° = −2.0 kcal/mol (30). Boryskina and coworkers (31) used a wide range of different experimental strategies to evaluate this identical parameter, estimating the power of every amide–amide hydrogen bond inside a poly(GlyProPro) CRP as ΔG° = −1.8 kcal/mol and inside native collagen as ΔG° = −1.4 kcal/mol. Open in a separate window
Glycine Substitutions Quite a few collagen-related illnesses are related to mutations in each triple-helical and nontriple-helical domains of assorted collagens ( ). These illnesses have been reviewed intimately elsewhere (32) and should not mentioned extensively herein. The Gly residue within the XaaYaaGly repeat is invariant in pure collagen, and favorable substitutions are unknown in CRPs (33). A computational examine urged that changing the obligate Gly residues of collagen with d-alanine or d-serine would stabilize the triple helix (34) and thus that the Gly residues in collagen are surrogates for nonnatural d-amino acids. Subsequent experimental knowledge demonstrated, nonetheless, that this notion was misguided (35). Lots of the most damaging mutations to collagen genes consequence within the substitution of a Gly residue concerned within the ladder of hydrogen bonds throughout the triple helix ( ). Each the identification of the amino acid changing Gly and the situation of that substitution can affect the pathology of, for instance, osteogenesis imperfecta (OI) (33, 36). Substitutions for Gly in proline-rich parts of the collagen sequence ( ) are far much less disruptive than these in proline-poor areas, a testomony to the significance of Professional derivatives for triple-helix nucleation (37). In vivo, triple helices fold in a C-terminal→N-terminal method (38). The time delay between disruption of triple-helix folding by a Gly substitution and renucleation of the folding course of N-terminal to the substitution web site is way shorter when triple-helix nucleating, proline-rich sequences are instantly N-terminal to the substitution web site (37). Any delay in triple-helix folding ends in overmodification of the protocollagen chains [in particular, inordinate hydroxylation of Lys residues N-terminal to the Gly substitution and excessive glycosylation of the resultant hydroxylysine residues ( )], thereby perturbing triple-helical construction and contributing to the severity of OI (39). Thus, the severity of OI correlates with the abundance of triple-helix nucleating, proline-rich sequences instantly N-terminal to the substitution web site (36).
Prolines within the Xaa and Yaa Positions Within the strands of human collagen, ~22% of all residues are both Professional or Hyp (9). The abundance of those residues preorganizes the person strands in a PPII conformation, thereby reducing the entropic price for collagen folding (40). Regardless of their stabilizing properties, Professional derivatives even have sure deleterious penalties for triple-helix folding and stability that partially offset their favorable results. For instance, Professional has a secondary amino group and kinds tertiary amides inside a peptide or protein. Tertiary amides have a major inhabitants of each the trans and the cis isomers ( ), whereas all peptide bonds in collagen are trans. Thus, earlier than a (ProHypGly) n strand can fold right into a triple helix, all of the cis peptide bonds should isomerize to trans. N-Methylalanine (an acyclic, tertiary amide lacking solely Cγ of Professional) decreases triple-helix stability when used to exchange Professional or Hyp in CRPs, presumably as a result of it lacks the preorganization imposed by the pyrrolidine ring of Professional derivatives (41). In distinction, avoiding the difficulty of cis-trans isomerization altogether by changing a Gly–Professional amide bond with a trans-locked alkene isostere additionally ends in a destabilized triple helix, regardless of leaving all interchain hydrogen bonds intact (42). Clearly, the elements dictating triple-helix construction and stability are intertwined in a fancy method (vide infra). Open in a separate window Professional residues within the Yaa place of protocollagen triplets are modified by prolyl 4-hydroxylase (P4H), a nonheme iron enzyme that catalyzes the posttranslational and stereoselective hydroxylation of the unactivated γ-carbon of Professional residues within the Yaa place of collagen sequences to kind Hyp ( ). P4H exercise is required for the viability of each the nematode Caenorhabditis elegans and the mouse Mus musculus (43, 44). Thus, Hyp is crucial for the formation of sound collagen in vivo. Open in a separate window
Proline Derivatives within the Xaa Place The Cγ-exo ring pucker of Professional residues within the Yaa place enhances triple-helix stability. Likewise, the ring pucker of Professional within the Xaa place is vital for triple-helix stability. Usually, Professional residues within the Xaa place of organic collagen should not hydroxylated and normally show the Cγ-endo ring pucker (67). By using Cγ-substituents, each the gauche impact and steric results might be availed to preorganize the Cγ-endo ring pucker ( and ). Set up of flp, (2S,4S)-4-chloroproline (clp), or (2S,4R)-4-methylproline (mep) residues (all of which want the Cγ-endo ring pucker) ( ) within the Xaa place of collagen is stabilizing relative to Professional, however set up of Flp, Clp, or Hyp (which want the Cγ-exo ring pucker) is destabilizing ( ) (61, 65, 68–70). These outcomes counsel that preorganizing the Cγ-endo ring pucker within the Xaa place of CRPs stabilizes triple helices. This conclusion is cheap as a result of Professional derivatives with a Cγ-endo ring-pucker have φ and ψ primary chain torsion angles just like these noticed within the Xaa place of triple helices ( ). Notably, changing Professional within the Xaa place of (ProProGly) 10 with hyp, a Professional spinoff that, like flp and clp, ought to want the Cγ-endo ring pucker owing to the gauche impact, yields CRPs that don’t kind triple helices ( ) (47). This anomalous consequence for hyp within the Xaa place may very well be attributable to deleterious hydration, idiosyncratic conformational preferences of hyp residues, or each (71). Kind IV collagen, which is the first part of basement membranes, has a excessive incidence of (2S,3S)-3-hydroxyproline (3S-Hyp) within the Xaa place (72). This modification is current in another collagen sorts and in invertebrate collagens. 3S-Hyp, which prefers a Cγ-endo ring pucker (73), is launched nearly completely inside ProHypGly triplets through posttranslational modification of particular person collagen strands by prolyl 3-hydroxylase (P3H), which is distinct from P4H (74). A recessive type of OI is related to a P3H deficiency (75, 76). Sure mutations to the gene encoding cartilage-associated protein, a P3H-helper protein, stop 3S-hydroxylation of α1(I)Pro986 in addition to 3S-hydroxylation of another Xaa-position Professional residues, leading to a phenotype practically equivalent to classical OI. The underlying foundation for the significance of 3S-hydroxylation of α1(I)Pro986 is unclear however may contain decrease charges of triple-helix secretion (76). Changing Professional with 3S-Hyp within the Xaa place of CRPs can improve triple-helix stability barely (73, 77). A crystal construction of a triple helix containing 3S-Hyp substitutions reveals the upkeep of the prototypical triple-helix construction and the absence of unfavorable steric interactions ( ) (78). In distinction, changing 3S-Hyp with (2S,3S)-3-fluoroproline destabilizes a triple helix markedly, presumably owing to a through-bond inductive impact that diminishes the flexibility of its primary chain oxygen to just accept a hydrogen bond ( ) (79).
An n→π* Interplay A normal precept within the design of CRPs is that Professional residues with both a Cγ-endo or Cγ-exo ring pucker will stabilize triple helices within the Xaa and Yaa positions, respectively ( – ). Acceptable ring pucker, enforced by a stereoelectronic or steric impact, preorganizes the φ and ψ torsion angles to these required for triple-helix formation. Intriguingly, the steadiness of a (flpProGly) 7 or (clpProGly) 10 triple helix is considerably lower than that of a (ProFlpGly) 7 or (ProClpGly) 10 triple helix, respectively ( ) (61, 68). Likewise, a (mepProGly) 7 triple helix is much less secure than a (ProMepGly) 7 triple helix ( ) (65). Two elements contribute to the decrease stability of triple helices shaped from CRPs with stabilizing Professional derivatives substituted within the Xaa place moderately than the Yaa place. First, a Cγ-endo ring pucker is already favored in Professional (56); flp, clp, and mep merely improve that desire ( ). In distinction, Flp, Clp, Hyp, and Mep have the extra dramatic impact of reversing the popular ring pucker of Professional, thereby assuaging the entropic penalty for triple-helix formation to a higher extent ( ). Second, Flp, Clp, and Mep within the Yaa place trigger favorable preorganization of all three primary chain torsion angles (φ, ψ, and ω) ( ). In distinction, flp, clp, and mep have a low chance of adopting a trans peptide bond (ω = 180°) (54, 61, 65) relative to Professional ( ), thereby mitigating the profit accrued from correct preorganization of φ and ψ. Notably, 13C-NMR research on collagen in vitro present that 16% of Gly–Professional bonds in unfolded collagen are within the cis conformation, whereas solely 8% of Xaa–Hyp bonds in unfolded collagen are cis, an statement that confirms the impact of Cγ-substitution on the conformation of the previous peptide bond (80). How does the impact of a 4-X substituent on Professional ring pucker affect the peptide bond isomerization equilibrium fixed (Ok trans/cis ) ( and and )? The reason stems from one other stereoelectronic impact: an n→π* interplay (56, 81). In an n→π* interplay, the oxygen of a peptide bond (O i−1 ) donates electron density from its lone pairs into the antibonding orbital of the carbonyl within the subsequent peptide bond (C i ′=O i ) ( c,d). The Cγ-exo ring pucker of a Professional residue supplies a extra favorable O i−1 ⋯C i ′=O i distance and angle for an n→π* interplay than does the Cγ-endo pucker (56). Importantly, Ok trans/cis for peptidyl prolyl amide bonds is set by the pyrrolidine ring pucker and isn’t usually affected by the identification of substituents within the 4-position of the pyrrolidine ring (82). As a result of an n→π* interplay can happen provided that the peptide bond containing O i−1 is trans, the n→π* interplay has an affect on the worth of Ok trans/cis for primary chains with applicable torsion angles ( ). Thus, imposing a Cγ-exo pucker on a pyrrolidine ring within the Yaa place of a CRP preorganizes not solely the φ and ψ angles for triple-helix formation, but in addition the ω angle. Certainly, a single n→π* interplay can stabilize the trans conformation by ΔG° = −0.7 kcal/mol (81, 83).
Hyp within the Xaa Place Within the Xaa place, a Professional residue with a Cγ-endo pucker usually stabilizes a triple helix, whereas one with a Cγ-exo pucker destabilizes a triple helix. For instance, (HypProGly) n triple helices are far much less secure than (ProProGly) n triple helices ( ) (84) as a result of Hyp prefers the Cγ-exo ring pucker and thus preorganizes the φ and ψ torsion angles improperly for the Xaa place of a collagen triple helix ( ). Surprisingly, (HypHypGly) 10 triple helices are literally barely extra secure than (ProHypGly) 10 triple helices ( ) (85, 86) regardless of the Hyp residues within the Xaa place of (HypHypGly) 10 displaying the Cγ-exo ring pucker within the triple helix (87, 88). It’s noteworthy that crystal constructions of (HypHypGly) 10 present that the principle chain torsion angles within the Xaa place of a (HypHypGly) n triple helix regulate to accommodate a Cγ-exo ring pucker in that place (87, 88). The discovering that Hyp can stabilize triple helices within the Xaa place in a context-dependent method was presaged in a examine by Gruskin and coworkers (89) on the worldwide substitution of Hyp for Professional in recombinant sort I collagen polypeptides that shaped secure triple helices. Notably, Hyp is discovered within the Xaa place of some invertebrate collagens (90) and might be acceptable in CRPs through which the Yaa place residue is just not Professional (86, 91, 92). Berisio and coworkers (93) have urged that (HypHypGly) 10 triple helices could be hyperstable owing to interstrand dipole-dipole interactions between proximal Cγ–OH bonds of adjoining Hyp residues. Kobayashi and coworkers (87) have proposed that the steadiness of (HypHypGly) 10 triple helices is attributable to the excessive hydration stage of the peptide chains within the single-coil state previous to triple-helix formation, which may cut back the entropic price of water bridge formation. A mixture of those elements is prone to be accountable for this anomaly.
Heterotrimeric Artificial Triple Helices Each flp and Flp enormously improve triple-helix stability when within the Xaa and Yaa place, respectively. Nonetheless, (flpFlpGly) n kinds a lot much less secure triple helices than does (ProProGly) n ( ) (79, 94). In such a helix, the fluorine atoms of flp and Flp residues in alternating strands could be proximal, and the C–F dipoles would work together unfavorably ( ) (79). These destructive steric and digital interactions presumably compromise triple-helix stability regardless of applicable preorganization of primary chain torsion angles. This speculation was confirmed by two different findings. First, a (clpClpGly) 10 triple helix doesn’t even kind at 4°C, whereas a (flpFlpGly) 10 triple helix has T m = 30°C ( ) (61, 94). The steric conflict between chlorine atoms of opposing clp and Clp residues is exacerbated by the big dimension of chlorine relative to fluorine ( ). Second, (mepMepGly) 7 kinds extra secure triple helices than do both of the corresponding mono-substituted CRPs, (mepProGly) 7 and (ProMepGly) 7 ( ). The 4-methyl teams protrude radially from the triple helix ( ) and thus can not work together detrimentally with one another (65). Open in a separate window The steric and stereoelectronic results on triple-helix stability manifested within the (flpFlpGly) 7 CRP supplied, for the primary time, a way to generate noncovalently linked, heterotrimeric triple helices with outlined stoichiometry. Evaluation of triple-helix cross sections urged a triple helix composed of (flpFlpGly) 7 :(ProProGly) 7 in both a 1:2 or 2:1 ratio may very well be secure, because the presence of some Professional residues within the Xaa and Yaa positions would remove deleterious steric interactions between fluorine residues in opposing strands. A (flpFlpGly) 7 :(ProProGly) 7 ratio of two:1 yielded probably the most secure triple helices, thereby demonstrating the primary occasion of heterotrimeric meeting of triple helices with managed stoichiometry (79) and suggesting the potential of creating a “code” for triple-helix meeting alongside the traces of the Watson-Crick code for DNA meeting. Gauba & Hartgerink (95) developed an alternate technique that employs Coulombic interactions to information the meeting of heterotrimeric triple helices. They noticed {that a} 1:1:1 combination of (ProArgGly) 10 :(GluHypGly) 10 :(ProHypGly) 10 produces triple helices containing one negatively charged, one positively charged, and one impartial CRP. Intriguingly, a (ProLysGly) 10 :(AspHypGly) 10 :(ProHypGly) 10 triple helix has a T m worth just like that of a (ProHypGly) 10 homotrimer, despite the fact that Asp and Lys are recognized to destabilize considerably the triple helix relative to Professional and Hyp ( ). This discovering demonstrates the utility of Coulombic interactions for stabilizing triple helices (96). Artificial collagen heterotrimers are interesting mimics of pure collagen strands, as most collagen sorts are themselves heterotrimers ( ). Gauba & Hartgerink (97) employed their Coulombic strategy to generate mimics of sort I collagen variants that result in OI. Particularly, they studied the steadiness of triple-helical heterotrimers containing one, two, or three Gly→Ser substitutions. They noticed {that a} Gly→Ser substitution in just one or two chains is just not as debilitating for triple-helix stability and folding as is a Gly→Ser substitution in all three chains.