Comprehensive analysis of protein folding activation thermodynamics reveals a universal behavior violated by kinetically stable proteases

Alpha-lytic protease (alpha LP) and Streptomyces griseus protease B (SGPB) are two extracellular serine proteases whose folding is absolutely dependent on the existence of their companion pro regions. Moreover, the native states of these proteins are, at best, marginally stable, with the apparent stability resulting from being kinetically trapped in the native state by large barriers to unfolding. Here, in an effort to understand the physical properties that distinguish kinetically and thermodynamically stable proteins, we study the temperature-dependences of the folding and unfolding kinetics of alpha LP and SGPB without their pro regions, and compare their behavior to a comprehensive set of other proteins. For the folding activation thermodynamics, we find some remarkable universal behaviors in the thermodynamically stable proteins that are violated dramatically by alpha LP. Despite significant variations in deltaC(P,F)++, the maximal folding speed occurs within the narrow biological temperature range for all proteins, except for alpha LP, with its maximal folding speed shifted lower by 200 K. This implies evolutionary pressures on folding speed for typical proteins, but not for alpha LP. In addition, the folding free energy barrier in the biological temperature range for most proteins is predominantly enthalpic, but purely entropic for alpha LP. The unfolding of alpha LP and SGPB is distinguished by three properties: a remarkably large deltaC(P,U)++, a very high deltaG(U)++, and a maximum deltaG(u)++ at the optimal growth temperature for the organism. While other proteins display each of these traits to some approximation, the simultaneous optimization of all three occurs only in the kinetically stable proteins, and appears to be required to maximize their unfolding cooperativity, by suppressing local unfolding events, and slowing the rate of global unfolding. Together, these properties extend the lifetime of these enzymes in the highly proteolytic extracellular environment. Attaining such functional properties seems possible only through the gross perturbation of the folding thermodynamics, which in turn has required the co-evolution of pro regions as folding catalysts.     

Quantifying optimal accuracy of local primary sequence bioinformatics methods

MOTIVATION: Traditional bioinformatics methods scan primary sequences for local patterns. It is important to assess how accurate local primary sequence methods can be. RESULTS: We study the problem of donor pre-mRNA splice site recognition, where the sequence overlaps between real and decoy datasets can be quantified, exposing the intrinsic limitations of the performance of local primary sequence methods. We assess the accuracy of primary sequence methods generally by studying how they scale with dataset size and demonstrate that our new primary sequence ranking methods have superior performance.     

The flexibility in the proline ring couples to the protein backbone

In proteins, the proline ring exists predominantly in two discrete states. However, there is also a small but significant amount of flexibility in the proline ring of high-resolution protein structures. We have found that this side-chain flexibility is coupled to the backbone conformation. To study this coupling, we have developed a model that is simply based on geometric and steric factors and not on energetics. We show that the coupling between phi and chi1 torsions in the proline ring can be described by an analytic equation that was developed by Bricard in 1897, and we describe a computer algorithm that implements the equation. The model predicts the observed coupling very well. The strain in the C(gamma)-C(delta)-N angle appears to be the principal barrier between the UP and DOWN pucker. This strain is relaxed to allow the proline ring to flatten in the rare PLANAR conformation.     

Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase

Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed in the absence of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa). However, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phospholipid was poor with a K(i) values (approximately 400 nM) that were approximately 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM). These results indicated that a significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain (Bajaj et al. (2001) J. Biol. Chem. 276, 16302-16309). Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII/Asp712Ala exhibited an 8-fold increased K(d) (35 +/- 1.5 nM) relative to wild-type suggesting a contribution by this residue of approximately 10% of the FVIIIa-FIXa binding energy. Thus both A2 and A3-C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.     

Fast-folding protein kinetics,hidden intermediates,and the sequential stabilization model

Do two‐state proteins fold by pathways or funnels? Native‐state hydrogen exchange experiments show discrete nonnative structures in equilibrium with the native state. These could be called hidden intermediates (HI) because their populations are small at equilibrium, and they are not detected in kinetic experiments. HIs have been invoked as disproof of funnel models, because funnel pictures appear to indicate (1) no specific sequences of events in folding; (2) a continuum, rather than a discrete ladder, of structures; and (3) smooth landscapes. In the present study, we solve the exact dynamics of a simple model. We find, instead, that the present microscopic model is indeed consistent with HIs and transition states, but such states occur in parallel, rather than along the single pathway predicted by the sequential stabilization model. At the microscopic level, we observe a huge multiplicity of trajectories. But at the macroscopic level, we observe two pathways of specific sequences of events that are relatively traditional except that they are in parallel, so there is not a single reaction coordinate. Using singular value decomposition, we show an accurate representation of the shapes of the model energy landscapes. They are highly complex funnels.     

Using motion planning to map protein folding landscapes and analyze folding kinetics of known native structures.

We investigate a novel approach for studying the kinetics of protein folding. Our framework has evolved from robotics motion planning techniques called probabilistic roadmap methods (PRMs) that have been applied in many diverse fields with great success. In our previous work, we presented our PRM-based technique and obtained encouraging results studying protein folding pathways for several small proteins. In this paper, we describe how our motion planning framework can be used to study protein folding kinetics. In particular, we present a refined version of our PRM-based framework and describe how it can be used to produce potential energy landscapes, free energy landscapes, and many folding pathways all from a single roadmap which is computed in a few hours on a desktop PC. Results are presented for 14 proteins. Our ability to produce large sets of unrelated folding pathways may potentially provide crucial insight into some aspects of folding kinetics, such as proteins that exhibit both two-state and three-state kinetics that are not captured by other theoretical techniques.     

Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.     

13C-nuclear magnetic resonance study of glycophorins AM and AN modified with various pyrylium salts

The environment of the N-terminal amino groups of glycophorins A^M and A^N has been studied using¹³C-NMR spectroscopy and pyrylium salts as amino-blocking agents. The extent of amino blocking was monitored by¹³C-reductive methylation of the residual free amino groups. The pyrylium ions reacted with the N-terminal amino groups of the two glycophorins at almost identical rates, which is thought to indicate that the overriding steric bulk of the pyrylium salt may determine the rate of the reaction. The difference in the rates of modification of lysine residues of glycophorins A^M and A^N by the pyrylium ions did indicate that there may exist an environmental difference around the lysine residues between the two glycophorins. This environmental difference may result from solution aggregation of the glycophorin A molecules or from some differences in the pK_a values of the five lysine residues found in glycophorins A^M and A^N.     

Can enzymes adopt a self-inhibited form? Results of x-ray crystallographic studies of chymosin.

Chymosin molecules in the crystal lattice have Tyr77 occluding the S1/S3 substrate binding pockets suggesting that the enzyme is self-inhibited. An analysis of this structure in conjunction with its comparison with pepsin has shown that this is most probably an intrinsic property of the enzyme. It also indicates that chymosin's substrate specificity may be dependent upon the ability of the substrate to displace the tyrosine ring from the binding pockets. This analysis also implies that active and self-inhibited forms of other aspartic proteinases can exist in solution helping to explain the results of kinetic studies of these enzymes.