Conformational origin of the aggregation of recombinant human factor VIII.

Aggregation of proteins is a major problem in their use as drugs and is also involved in a variety of pathological diseases. In this study, biophysical techniques were employed to investigate aggregate formation in the pharmaceutically important protein, recombinant human factor VIII (rhFVIII). Recombinant human factor VIII incubated in solution at 37 degrees C formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did not, however, suggest significant differences in protein conformation. To identify conformational changes in rhFVIII that may be involved in rhFVIII aggregation, temperature and solutes were used to perturb the native structure of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temperature revealed conformational changes corresponding to an increase in intermolecular beta-sheet content beginning at approximately 45 degrees C with significant aggregation observed above 60 degrees C. Fluorescence and DSC studies of rhFVIII also indicated conformational changes initiating between 45 and 50 degrees C. An increase in the exposure of hydrophobic surfaces was observed beginning at approximately 40 degrees C, as monitored by increased binding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis-ANS). Perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, characterized by an increase in the wavelength of the fluorescence emission maximum of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII. Moreover, this transition was correlated with an increased association of factor VIII upon incubation at 37 degrees C in the presence of various solutes. These results suggest that association of rhFVIII in solution was initiated by a small transition in the tertiary structure of the protein which produced a nucleating species that led to the formation of inactive soluble aggregates.

The basis for the super-repressor phenotypes of the AV77 and EK18 mutants of trp repressor.

The DNA-binding properties of two super-repressor mutants of the Escherichia coli trp repressor, EK18 and AV77, have been investigated using steady-state fluorescence anisotropy measurements, in order to further elucidate the basis for their super-repressor phenotypes. Several suggestions have been previously proposed as the basis for the super-repressor phenotype of EK18 and AV77. For the negative to positive charge change EK18 mutant, increased electrostatic interactions between the EK18 mutant and the operator and increased protein-protein interactions between EK18 dimers have been suggested as contributing to the super-repressor phenotype of this mutant. We show that EK18 dimers actually bind to wild-type and variant operator sequences with a decrease in apparent cooperativity and an increase in affinity, compared to WTTR dimers. Thus, the EK18 super-repressor phenotype is not due to increased cooperative binding between EK18 dimers. These results support the hypothesis that the super-repressor phenotype of EK18 arises from increased electrostatic interactions between the mutant and DNA. In the case of the AV77 mutant, weaker binding affinity of apo-AV77 to non-specific DNA, increased selectivity of binding of AV77 for the operator, and a higher population of folded functional AV77 dimers available to bind the operator under limiting L-Trp conditions in vivo, have been proposed for the super-repressor phenotype of this mutant. We show that like the EK18 mutant, apoAV77 binds with higher affinity to non-specific DNA compared to apo-WTTR and that the holo-AV77 mutant does not bind with higher selectivity to the operator, has had been previously proposed. We therefore conclude that the super-repressor phenotype of the AV77 mutant is due to an increase in the population of folded, functional AV77 dimers, under limiting L-Trp conditions in vivo.

Probing the role of water in the tryptophan repressor-operator complex.

The Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment. In the co-crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface. To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined. In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation. The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding. Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA. However, the complexity of the effects of betaine and the small or negligible effects of the other osmolytes could also arise from osmolyte induced competition between antagonistic coupled reactions.

Probing the physical basis for trp repressor-operator recognition.

The bacterial repressor protein, trp repressor, is one of the best studied transcriptional regulatory proteins in terms of function, structure, dynamics and stability. Despite these significant advances, the structural and energetic basis for the specific recognition of its operator sites by trp repressor remains poorly understood. In fact, recognition in this system is controled by the binding of the co-repressor ligand, l-tryptophan, as well as by conformational and dynamic properties of the operator targets, DNA sequence-dependent control of the oligomerization properties of the repressor, water-mediated interactions, and specific interactions involving the peptide backbone and phosphate moieties. Moreover, only one direct contact between the protein and the DNA is evident from the crystallographically determined structure of the complex. In an attempt to better define how the various sequence elements in the operator target contribute to this complex control of affinity and cooperativity of trp repressor binding, we have studied the binding of trp repressor to a series of mutated operator targets using fluorescence anisotropy, which provides very high quality data allowing fairly precise estimations of the affinities involved. We conclude from these studies that even on very small (25 bp) targets, the repressor binds slightly cooperatively, populating a 2:1 dimer/DNA complex, and then at higher concentrations a third dimer is bound with significantly lower affinity, revealing an inherent asymmetry in the trpEDCBA-derived target. Investigation of the basis for the asymmetry implicates the identity of the second base in the so-called structural half-site GNACT, which apparently influences the switch between tandem and simple binding. Mutation of the C or the T bases in the structural half-site abolishes all specificity in binding, and alteration of the single direct contact, the G of the structural half-site, or the central TTAA significantly lowers the affinity of the dimer for its site, without modifying the apparent cooperativity. Finally, we note that the order of affinity is conserved in the absence of the co-repressor, and moreover, it is in all cases significantly higher than that observed for holo-repressor binding to non-specific DNA, indicating that one cannot simply equate apo-repressor and non-specific binding.