A transient expansion of the native state precedes aggregation of recombinant human interferon-γ

Abstract

Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-␥ (rhIFN-␥) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-␥ aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-␥ and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-␥ native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-␥ against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.Formation of biologically inactive proteins by aggregation is a problem of considerable importance in many disciplines (1-3). For example, protein aggregates can be formed in vivo and in vitro during folding of nascent polypeptide chains, eliminating or reducing the protein's biological function (4, 5). Misfolded protein aggregates often can be observed as inclusion bodies (6, 7) and are implicated in amyloid deposition in vivo (8). In the biotechnology industry, protein aggregation is encountered routinely during purification, refolding, sterilization, shipping, and storage processes because of the presence of chemical, physical, and thermal stresses (9).In addition to significant losses in protein activity, clinical dangers result from parenteral administration of aggregated material (10). Even aggregation levels as low as 1% over a 2-year shelf life can render a product clinically unacceptable. Thus, proteins must be protected against even relatively mild stresses by the addition of proper excipients. Rational choice of these excipients requires insight into the mechanism of aggregation.The detailed mechanism of protein aggregation is still unclear. Usually, the aggregation pathway is modeled as shown in Scheme 1 by using the Lumry-Erying framework (11,12). The model involves a first-order reversible unfolding of the protein and subsequent aggregation of nonnative species in a higher order process (2, 11-13):In this scheme, N refers to native protein, and A refers to an intermediate conformational state preceding aggregation. A m refers to an aggregated form composed of m pro...

Thermodynamic Modulation of Light Chain Amyloid Fibril Formation

“…From a state equilibrium viewpoint, as has previously been suggested for amyloid-forming variants of lysozyme, BIF at any instant in time has a greater fraction of partially folded species in its molecular population than does GAL (13,25,26). It is well established that non-native protein aggregates are formed from partially folded molecules, which are sparsely and transiently populated relative to the most compact native conformation with which they are in equilibrium (13,(27)(28)(29)(30)(31). With the relatively unstable amyloidogenic protein variants, there are sufficiently high levels of partially folded molecules that, during the time scale for amyloid fibril formation of days to weeks or longer, fibrils can form even in nonperturbing environments.…”

“…In addition to increasing the free energy barrier between native and unfolded states (14,15), sucrose also shifts the equilibrium between protein states toward the most compact native conformation and away from expanded species and, hence, reduces the H-D exchange rate (16,27,28). With BIF, 1.0 M sucrose reduced the rate of exchange to approximately that noted with nonpathological GAL in PBS alone (Fig.…”

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

“…However, even at temperatures well below the melting temperature, thermal fluctuations are expected to transiently expose the hydrophobic interior to the solvent while keeping structural elements intact, and this may prime aggregation. 12 In fact, even subtle changes to the protein conformation may trigger the prevalence of attractive intermolecular forces. 3 Aggregation is hence most likely initiated by minor structural changes to certain parts of the molecule and possibly to just a small fraction of the sample.…”

“…10,11 Due to minute transient perturbations of the molecular structure, heating may induce aggregation even well below the melting temperature of the protein. 1,12 Furthermore, higher temperature accelerates aggregation because of its impact on the individual rate constants involved in the aggregation kinetics. This is the reason why high temperature is often used to probe the stability of a given protein.…”

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

“…For example, we have recently shown (48,49) that the surface area and volume changes required to form an aggregation competent species of recombinant human interferon-␥ corresponds to that expected for formation of a molten globule intermediate. Because aggregation may be inhibited under pressure (25), eliminating interference from aggregate signals, we suggest that high pressure spectroscopy may be of particular value for structural studies of molten globules induced either by high pressures or solution composition.…”