Two proteins with the same structure respond very differently to mutation: the role of plasticity in protein stability.

As part of a systematic study of the folding of protein structural families we compare the effect of mutation in two closely related fibronectin type III (fnIII) domains, the tenth fnIII domain of human fibronectin (FNfn10) and the third fnIII domain of human tenascin (TNfn3). This comparison of the two related proteins allows us to distinguish any anomalous response to mutation. Although they have very similar structures, the effect of mutation is very different. TNfn3 behaves like a "typical" protein, with changes in free energy correlated to the number of contacts lost on mutation. The loss of free energy upon mutation is significantly lower for FNfn10, particularly mutations of residues in the A, B and G strands. Remarkably, some of the residues involved are completely buried and closely packed in the core. In FNfn10 the regions of the protein that can accommodate mutation have previously been shown to be mobile. We propose that there is a "plasticity" in the peripheral regions of FNfn10 that allows it to rearrange to minimise the effect of mutations. This study emphasises the difficulties that might arise when making generalisations from a single member of a protein family.

The folding of an immunoglobulin-like Greek key protein is defined by a common-core nucleus and regions constrained by topology.

TNfn3, the third fibronectin type III domain of human tenascin, is an immunoglobulin-like protein that is a good model for experimental and theoretical analyses of Greek key folding. The third fibronectin type III domain of human tenascin folds and unfolds in a two-state fashion over a range of temperature and pH values, and in the presence of stabilising salts. Here, we present a high resolution protein engineering analysis of the single rate determining transition state. The 48 mutations report on the contribution of side-chains at 32 sites in the core and loop regions. Three areas in the protein exhibit high Phi-values, indicating that they are partially structured in the transition state. First, a common-core ring of four positions in the central strands B, C, E and F, that are in close contact, form a nucleus of tertiary interactions. The two other regions that appear well-formed are the C' region and the E-F loop. The Phi-values gradually decrease away from these regions such that the very ends of the two terminal strands A and G, have Phi-values of zero. We propose a model for the folding of immunoglobulin-like proteins in which the common-core "ring" forms the nucleus for folding, whilst the C' and E-F regions are constrained by topology to pack early. Folding characteristics of a group of structurally related proteins appear to support this model.

Conservation of folding and stability within a protein family: the tyrosine corner as an evolutionary cul-de-sac.

What are the selective pressures on protein sequences during evolution? Amino acid residues may be highly conserved for functional or structural (stability) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are using an experimental "fold approach" to the study of protein folding. This compares the folding and stability of a number of proteins that share the same fold, but have no common amino acid sequence or biological activity. The fold selected for this study is the immunoglobulin-like beta-sandwich fold, which is a fold that has no specifically conserved function. Four model proteins are used from two distinct superfamilies that share the immunoglobulin-like fold, the fibronectin type III and immunoglobulin superfamilies. Here, the fold approach and protein engineering are used to question the role of a highly conserved tyrosine in the "tyrosine corner" motif that is found ubiquitously and exclusively in Greek key proteins. In the four model beta-sandwich proteins characterised here, the tyrosine is the only residue that is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucleate folding in the immunoglobulin superfamily, whereas it is involved to some extent in early structure formation in the fibronectin type III superfamily. The tyrosine corner is important for stability, mutation to phenylalanine costs between 1.5 and 3 kcal mol(-1). We propose that the high level of conservation of the tyrosine is related to the structural restraints of the loop connecting the beta-sheets, representing an evolutionary "cul-de-sac".

Folding studies of immunoglobulin-like beta-sandwich proteins suggest that they share a common folding pathway.

BACKGROUND: Are folding pathways conserved in protein families? To test this explicitly and ask to what extent structure specifies folding pathways requires comparison of proteins with a common fold. Our strategy is to choose members of a highly diverse protein family with no conservation of function and little or no sequence identity, but with structures that are essentially the same. The immunoglobulin-like fold is one of the most common structural families, and is subdivided into superfamilies with no detectable evolutionary or functional relationship. RESULTS: We compared the folding of a number of immunoglobulin-like proteins that have a common structural core and found a strong correlation between folding rate and stability. The results suggest that the folding pathways of these immunoglobulin-like proteins share common features. CONCLUSIONS: This study is the first to compare the folding of structurally related proteins that are members of different superfamilies. The most likely explanation for the results is that interactions that are important in defining the structure of immunoglobulin-like proteins are also used to guide folding.

The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease.

Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions. Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a beta-sandwich fold. Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.

The dependence of chemical exchange on boundary selection in a fibronectin type III domain from human tenascin.

The third fibronectin type III domain from human tenascin adopts a compact beta-sandwich fold. Its boundaries were originally selected to encode a 90-residue domain (TNfn31-90). We conclude that the dynamic properties of TNfn3 are more accurately represented when the C terminus is extended by the two naturally succeeding residues. Longitudinal (R1) and transverse (R2) 15N relaxation rates, and ¿1H-15N¿ NOE enhancements at pH 4.9 and 300 K are presented for TNfn31-90 and TNfn31-92, the extended form, at two field strengths (11.74 and 14.10 T). Nearly identical results confirm their similar motional properties over a broad range of timescales. However, a number of residues near the C terminus in TNfn31-90 exhibit elevated transverse relaxation rates and broadened signals in 1H-15N HSQC spectra. Explicit rates of chemical exchange for five residues in TNfn31-90 were determined by measuring transverse relaxation rates in a series of CPMG experiments with spin-echo refocusing delays increasing from 311 to 1436 micros. Calculated exchange rates average 1000(+/-311) s-1, with individual uncertainties near 20%. Homonuclear TOCSY experiments collected between pH 4 and 7 reveal the coincident titration of two acidic clusters in TNfn31-90 at pH 5. 64(+/-0.47). The repulsive electrostatic interaction of the C-terminal carboxylate with one of these clusters may promote chemical exchange in the shorter domain. Additionally, NOE and chemical shift data suggest hydrogen bond formation between the added residues and adjacent loops. The data affirm the importance of judiciously selecting domain boundaries prior to the characterization of molecular properties.

Dual recognition and the role of specificity-determining residues in colicin E9 DNase-immunity protein interactions.

The immunity protein Im2 can bind and inhibit the noncognate endonuclease domain of the bacterial toxin colicin E9 with a Kd of 19 nM, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 with which it shares 68% sequence identity. Previous work from our laboratory has shown that the specificity differences of these four-helix immunity proteins is due almost entirely to helix II which is largely variable in sequence in the immunity protein family. From alanine scanning mutagenesis of Im9 in conjunction with high-field NMR data, a dual recognition model for colicin-immunity protein specificity has been proposed whereby the conserved residues of helix III of the immunity protein act as the anchor of the endonuclease binding site while the variable residues of helix II control the specificity of the protein-protein interaction. In this work, we identify three residues (at positions 33, 34, and 38) in helix II which define the specificity differences of Im2 and Im9 for colicin E9 and, using alanine mutagenesis of the putative endonuclease binding surface of Im2, compare the distribution of binding energies for conserved and nonconserved sites in both immunity proteins. This comparison highlights the conserved residues of both Im2 and Im9 as the major determinants of E9 DNase binding energy. Conversely, the nonconserved, specificity-determining residues only contribute to the E9 DNase binding energy in the cognate Im9 protein, while in the noncognate immunity protein Im2, they either destabilize the complex or do not contribute to the binding energy. This comparative alanine scan of two immunity proteins therefore supports the dual recognition mechanism of selectivity in colicin-immunity protein interactions and provides a basis for understanding specificity in other protein-protein interaction systems involving structurally conserved protein families.

The effect of boundary selection on the stability and folding of the third fibronectin type III domain from human tenascin.

Correct selection of domain boundaries is critical for structural analysis of single domains from multimodular proteins. Folding and stability studies of the third fibronectin type III domain from human tenascin (TNfn31-90) have shown that it is moderately stable (Delta G(D-N)(H2O) approximately 5 kcal mol-1) and folds with two-state kinetics. In an attempt to stabilize the protein, five domains were constructed with different combinations of extensions to the N- and C-termini. Thermal denaturation studies show that a specific two amino acid (Gly-Leu) extension at the C-terminus is primarily responsible for a significant increase in stability. The Delta Delta G(D-N)(H2O) of the Gly-Leu extension (TNfn3(1-92)) is 2.7 +/- 0.3 kcal mol-1. Refolding kinetics do not differ significantly, but unfolding is slowed 40-fold. Mutation of leucine 92 to alanine does not affect stability, indicating that the stability of the extension does not come from the packing of the leucine side chain. Hydrogen exchange data suggest that the extension adds new hydrogen bonds and strengthens existing hydrogen bonds in the C-terminal interaction with the A-B and E-F loops. Removal of a very small number of hydrogen bonds substantially increases the unfolding rate, a phenomenon which may be important in stress-relaxation of FNIII-containing muscle proteins such as titin. These experiments demonstrate the importance of a small number of additional long-range interactions in the overall formation of a compact independently folding beta-sheet module.

Folding and stability of a fibronectin type III domain of human tenascin.

The folding of an isolated fibronectin type III domain of human tenascin, a large extra-cellular matrix protein, has been characterised. The isolated module, which has no disulphide bonds, can be reversibly unfolded by chemical denaturant and temperature. Equilibrium unfolding, measured using a number of different probes, fits to a two-state transition, with consistent measures of DeltaGH2OD-N. Folding and refolding rate constants have been determined over a range of denaturant concentrations. The refolding kinetics are bi-phasic, and in the transition region the slow phase dominates refolding kinetics. Outside the transition region the folding of the fast-folding species fits to a two-state model. There is no evidence for significant accumulation of partially folded intermediates.

Structure and stability of an immunoglobulin superfamily domain from twitchin, a muscle protein of the nematode Caenorhabditis elegans.

The NMR solution structure of an immunoglobulin superfamily module of twitchin (Ig 18') has been determined and the kinetic and equilibrium folding behaviour characterised. Thirty molecular coordinates were calculated using a hybrid distance geometry-simulated annealing protocol based on 1207 distance and 48 dihedral restraints. The atomic rms distributions about the mean coordinate for the ensemble of structures is 0.55( +/- 0.09) A for backbone atoms and 1.10( +/- 0.08) A for all heavy atoms. The protein has a topology very similar to that of telokin and the titin Ig domains and thus it falls into the I set of the immunoglobulin superfamily. The close agreement between the predicted and observed structures of Ig 18' demonstrates clearly that the I set profile can be applied in the structure prediction of immunoglobulin-like domains of diverse modular proteins. Folding studies reveal that the protein has relatively low thermodynamic stability, deltaG(H2O)U-F = 4.0 kcal mol(-1) at physiological pH. Unfolding studies suggest that the protein has considerable kinetic stability, the half life of the unfolding is greater than 40 minutes in the absence of denaturant.

Effects of antihypertensive therapy on blood pressure control, cognition, and reactivity. A placebo-controlled comparison of prazosin, propranolol, and hydrochlorothiazide.

A randomized, placebo-controlled trial was conducted to compare the effects of treatment with prazosin, propranolol, or hydrochlorothiazide on the following variables: blood pressure, cognitive and psychomotor skills, cardiovascular reactivity to natural and laboratory challenges, and serum lipid and lipoprotein levels. Side effects were recorded and patients evaluated how they felt during their treatment. Sixty-nine men, 35 percent black, aged 25 to 55 (mean 51.3) years, with diastolic blood pressures between 90 and 104 mm Hg (mean, 93.3 mm Hg), completed the study. There were no differences between active treatment groups in the proportion of patients with controlled blood pressure during the maintenance phase of the study. In the cognitive and psychomotor tests, the hydrochlorothiazide group showed significantly less improvement from baseline than the other treatment groups on the block design subscale of the Wechsler Adult Intelligence Scale-Revised, and there was a trend for the propranolol group to have less improvement from baseline than the other groups on the digit span subscale. There were no other significant pretreatment to post-treatment changes in the other cognitive or psychomotor tests, the Russell Revision of the Wechsler Memory Scale, or a number of computerized reaction-time and signal-detection tasks. In the reactivity testing, there was a significantly lower increase in heart rate in the prazosin group compared with placebo during the second laboratory challenge of the Stroop Color Interference Test. Post-treatment declines in ambulatory blood pressure were seen in all of the drug treatment groups in average and maximal diastolic and systolic blood pressures. Both propranolol and hydrochlorothiazide treatment resulted in low-density lipoprotein cholesterol levels that were higher than baseline and the hydrochlorothiazide treatment had significantly increased total cholesterol levels. In contrast, the prazosin-treated group experienced no adverse changes in these parameters. Overall, the propranolol group had significantly more moderate and severe side effects than did the other three groups. Considering the pattern of blood pressure control, cognitive and psychomotor effects, changes in lipid levels, and magnitude of side effects, prazosin seems to have the most advantageous profile in this study of the three anti-hypertensive agents evaluated.

A kinetic study comparing the light-reversal properties of carbon monoxide inhibition of ethylmorphine, benzphetamine, and 7-ethoxycoumarin dealkylation with those of hydroxylation of 17-hydroxyprogesterone and testosterone.

The light-reversal properties of carbon monoxide (CO) inhibition of the dealkylation of benzphetamine, ethylmorphine, and 7-ethoxycoumarin by microsomes from phenobarbital (PB)-induced rat livers were compared with those of the 6 beta-, 7 alpha-, and 16 alpha-hydroxylations of testosterone by the same rat hepatic microsomes and C-21 hydroxylation of 17-OH progesterone by steer adrenal microsomes. CO inhibited all reactions studied to essentially the same degree. The significant finding was that the dealkylations were reversed most effectively by light of wavelengths between 440 and 445 nm, rather than around 450 nm, the optimal wavelength for steroid hydroxylations. Moreover, the dealkylations required several-fold higher light intensities for equivalent light reversal. These studies suggest that the heme protein-CO complex responsible for dealkylations has a spectrum corresponding to the shape of the pass band of the 445-nm filter, whereas that of the steroid hydroxylations has its light-reversal maximum at 450 nm and appears to be broader. The measurable differences in the light-reversal properties between the monooxygenations of two groups of substrates, (i) dealkylations and (ii) hydroxylations of lipid substrates, furnish biophysical properties that allow a better characterization of microsomal monooxygenases which should be of value in forwarding progress in the study of these systems.

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres.

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree inhibition of the demethylation reactions by a variety of CO:O2 mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O2 utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.