natural selection ͉ homologous proteins ͉ structural pK shifts ͉ conformational ensembles ͉ differential scanning calorimetry C onventionally, the folding and function of single-domain proteins are described as two-state processes in analogy to elementary chemical reactions. The protein molecule is assumed to reside in either of two states: folded or active and unfolded or inactive, which interconvert by crossing a high free-energy barrier with transition-state-like kinetics. However, proteins can in principle exist in many different conformations or microstates because of their thousands of degrees of freedom. Such inherent complexity is best described by using low-dimensional freeenergy surfaces, which are obtained by projecting the solventaveraged energy as a function of atomic coordinates onto a few order parameters [the energy landscape approach (1)]. A freeenergy surface description includes the two-state folding model as a particular scenario, but is more general. In this approach barriers, basins of attraction, and even finer topographical details of the surface (i.e., roughness) emerge from detailed balancing between conformational entropy and the energy from stabilizing interactions (see refs. 2 and 3 for some specific examples). Therefore, surfaces that exhibit marginal barriers or are even completely barrierless appear as interesting alternatives to the two-state folding picture (1). These predictions have been confirmed experimentally in recent years (4-6).If the folding barriers are comparable to the thermal energy (RT), ensembles of conformations with an intermediate degree of structure become significantly populated and interconvert with diffusive dynamics. This realization opens a realm of possibilities for the experimental study of protein folding reactions and mechanisms (recently reviewed in ref. 7). Moreover, it also has important implications for protein function because the conformational fluctuations associated with marginal folding barriers could be exploited to modulate activity and/or synchronize the action of enzymes in sequential reactions (8). Examples of how this could be achieved have been recently explored for protein binding with the fly-cast model (9, 10) and related analyses (11), as well as for the optimization of allosteric coupling (12).Another important consequence of shallow free-energy surfaces is that their shape can be resolved in equilibrium experiments sensitive to the energy fluctuations associated with protein conformation, such as differential scanning calorimetry (DSC). Building on this idea, Muñoz and Sanchez-Ruiz have recently developed a phenomenological variable-barrier model for the analysis of DSC data (13). In this analysis, the DSC thermogram is fitted to an idealized (i.e., a Landau quartic polynomial) one-dimensional free-energy surface from which it is possible to estimate the barrier height and the population of conformational ensembles with an intermediate degree of structure (13). Although these folding barriers are intrinsically ''thermodynamic,'' ...
Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.
The wide range of metabolic phenotypes in phenylketonuria is due to a large number of variants causing variable impairment in phenylalanine hydroxylase function. A total of 834 phenylalanine hydroxylase gene variants from the locus-specific database PAHvdb and genotypes of 4181 phenylketonuria patients from the BIOPKU database were characterized using FoldX, SIFT Blink, Polyphen-2 and SNPs3D algorithms. Obtained data was correlated with residual enzyme activity, patients' phenotype and tetrahydrobiopterin responsiveness. A descriptive analysis of both databases was compiled and an interactive viewer in PAHvdb database was implemented for structure visualization of missense variants. We found a quantitative relationship between phenylalanine hydroxylase protein stability and enzyme activity (r s ¼ 0.479), between protein stability and allelic phenotype (r s ¼ À0.458), as well as between enzyme activity and allelic phenotype (r s ¼ 0.799). Enzyme stability algorithms (FoldX and SNPs3D), allelic phenotype and enzyme activity were most powerful to predict patients' phenotype and tetrahydrobiopterin response. Phenotype prediction was most accurate in deleterious genotypes (E100%), followed by homozygous (92.9%), hemizygous (94.8%), and compound heterozygous genotypes (77.9%), while tetrahydrobiopterin response was correctly predicted in 71.0% of all cases. To our knowledge this is the largest study using algorithms for the prediction of patients' phenotype and tetrahydrobiopterin responsiveness in phenylketonuria patients, using data from the locus-specific and genotypes database.
Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.
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