Effect of pegylation on self-association of IFN-α2b.

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-α2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-α2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-α2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.

Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: sequence analysis and structural studies on triple-helix peptide models.

Interruptions in the repeating (Gly-X1-X2)(n) amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher-order structure and function. This study defines the sequence features and structural consequences of the most common interruption, where one residue is missing from the tripeptide pattern, Gly-X1-X2-Gly-AA(1)-Gly-X1-X2, designated G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated G4G-type, in that both are destabilizing, but allow continuation of rod-like triple helices and maintenance of the single residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix, while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.

Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline.

The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (Gly-Xaa-X'aa)(79), which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (Gly-Xaa-X'aa)(n) sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36 degrees C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27 degrees C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.

Self-association of collagen triple helic peptides into higher order structures.

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as beta-amyloid formation. The triple helical form of (Pro-Hyp-Gly)(10), a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleation-growth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25 degrees C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (Pro-Hyp-Gly)(10) forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.

Conformational features of a natural break in the type IV collagen Gly-X-Y repeat.

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.

Stability junction at a common mutation site in the collagenous domain of the mannose binding lectin.

Missense mutations in the collagen triple-helix that replace one of the required Gly residues in the (Gly-Xaa-Yaa)(n)() repeating sequence have been implicated in various disorders. Although most hereditary collagen disorders are rare, a common occurrence of a Gly replacement mutation is found in the collagenous domain of mannose binding lectin (MBL). A Gly --> Asp mutation at position 54 in MBL is found at a frequency as high as 30% in certain populations and leads to increased susceptibility to infections. The structural and energetic consequences of this mutation are investigated by comparing a triple-helical peptide containing the N-terminal Gly-X-Y units of MBL with the homologous peptide containing the Gly to Asp replacement. The mutation leads to a loss of triple-helix content but only a small decrease in the stability of the triple-helix (DeltaT(m) approximately 2 degrees C) and no change in the calorimetric enthalpy. NMR studies on specifically labeled residues indicate the portion of the peptide C-terminal to residue 54 is in a highly ordered triple-helix in both peptides, while residues N-terminal to the mutation site have a weak triple-helical signal in the parent peptide and are completely disordered in the mutant peptide. These results suggest that the N-terminal triplet residues are contributing little to the stability of this peptide, a hypothesis confirmed by the stability and enthalpy of shorter peptides containing only the region C-terminal to the mutation site. The Gly to Asp replacement at position 54 in MBL occurs at the boundary of a highly stable triple-helix region and a very unstable sequence. The junctional position of this mutation minimizes its destabilizing effect, in contrast with the significant destabilization seen for Gly replacements in peptides modeling collagen diseases.