Understanding the initial steps of protein aggregation leading to the formation of amyloid fibrils remains a challenge. Here, the kinetics of such a process is determined for a misfolding protein model, ADA2h. The double nature of the very early kinetics suggests a step model of aggregation, where the denatured polypeptide folds into an aggregated β-intermediate that subsequently reorganises into a more organised β-sheet-richer structure that finally results in amyloid fibre formation. To determine the regions of the protein involved in amyloidosis, we have analysed a series of mutants previously made to study ADA2h folding. Using the algorithm TANGO, we have designed mutants that should enhance or decrease aggregation. Experimental analysis of the mutants shows that the C terminus of the molecule (comprising the last and edge β-strand) is the major contributor to amyloid fibril formation, in good agreement with theoretical predictions. Comparison with proteins with similar topology reveals that family folds do not necessarily share the same principles of protein folding and/or aggregation.

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.

The term 'amyloidosis' is used to represent a group of protein misfolding diseases characterized by the polymerization of normally innocuous and soluble proteins or peptides into insoluble proteinaceous deposits. One of the several questions that remain unclear regarding the process of amyloid fibril formation is related to the status of the protein when such a process begins. Protein engineering is one of the selected approaches to study amyloidosis. Characterization of many variants of a protein can give information about why a soluble protein aggregates to form fibrils. In the present study, we report information on the conformational changes that precede the formation of fibrils, monitored by the complementary use of exoproteolysis and matrix-assisted laser-desorption ionization-time-of-flight-MS. This is a novel application of an easy and fast approach. In addition, we used it to evaluate the ability of the model protein ADA2h (activation domain of human procarboxypeptidase A2) and their mutants to generate amyloid fibrils. It could be a useful test to screen protein variants and to study to what extent some physicochemical parameters affect fibrillogenesis.

The activation domain of human procarboxypeptidase A2, ADA2h, is an 81‐residue globular domain released during the proteolytic activation of the proenzyme. The role of this and other similar domains as assistants of the correct folding of the enzyme is not fully understood. The folding pathway of ADA2h was characterized previously, and it was also observed that under certain conditions it may convert into amyloid fibrils in vitro. To gain insight into these processes, a detailed description of its three‐dimensional structure in aqueous solution is required so that eventual changes could be properly monitored. A complete assignment of the 1H and 15N resonances of ADA2h was performed, and the solution structure, as derived from a set of 1688 nonredundant constraints, is very well defined (pairwise backbone RMSD = 0.67 ± 0.17 Å for residues 10–80). The structure is composed of two antiparallel α‐helices comprising residues 19–32 and 58–69 packed on the same side of a three‐stranded β‐sheet spanning residues 10–15, 50–55, and 73–75. The global fold for the isolated human A2 activation domain is very similar to that of porcine carboxypeptidase B, as well as to the structure of the domain in the crystal of the intact human proenzyme. The observed structural differences relative to the intact human proenzyme are located at the interface between the activation domain and the enzyme and can be related with the activation mechanism. The backbone amide proton exchange behavior of ADA2h was also examined. The global free energy of unfolding obtained from exchange data of the most protected amide protons at pH 7.0 and 298K is 4.9 ± 0.3 kcal.mole−1, in good agreement with the values determined by thermal or denaturant unfolding studies.

In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability.

Zyme/protease M/neurosin/human kallikrein 6 (hK6) is a member of the human kallikrein family of trypsin-like serine proteinases and was originally identified as being down-regulated in metastatic breast and ovarian tumors when compared with corresponding primary tumors. Recent evidence suggests that hK6 may serve as a circulating tumor marker in ovarian cancers. In addition, it was described in the brain of Parkinson's disease and Alzheimer's disease patients, where it is implicated in amyloid precursor protein processing. It is thus a biomarker for these diseases. To examine the mechanism of activation of hK6, we have solved the structure of its proform, the first of a human kallikrein family member. The proenzyme displays a fold that exhibits chimeric features between those of trypsinogen and other family members. It lacks the characteristic “kallikrein loop” and forms the six disulfide bridges of trypsin. Pro-hK6 displays a completely closed specificity pocket and a unique conformation of the regions involved in structural rearrangements upon proteolytic cleavage activation. This points to a novel activation mechanism, which could be extrapolated to other human kallikreins.

The activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well-defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.

Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix-engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two alpha helices in order to increase their stability. At neutral and alkaline pH the three mutants, alpha-helix 1 (M1), alpha-helix 2 (M2) and alpha-helix 1 and alpha-helix 2 (DM), were more stable than the wild-type domain, in the order DM, M2, M1 and wild-type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water-exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild-type and M1 have a strong tendency to aggregate, giving rise to a beta conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third beta strands as well as part of alpha-helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 310 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.

The cDNA of human procarboxypeptidase A2 has been overexpressed in the methylotrophic yeast Pichia pastoris and secreted into the culture medium by means of the alpha-mating factor signal sequence, yielding a major protein of identical size and N-terminal sequence as the wild-type form, Two other forms containing the proenzyme have also been overexpressed: one of them resulted from an incomplete processing of the signal peptide, whereas the other was a glycosylated derivative. Recombinant procarboxypeptidase A2 was purified to homogeneity, and it was shown that its mature active form displays functional properties similar to those of the enzyme directly isolated from human pancreas, The overall yield was similar to 250 mg of proenzyme or 180 mg of mature enzyme/liter of cell culture. The proteolysis-promoted activation process of the recombinant proenzyme has been studied in detail. During maturation by trypsin, the increase in activity of the enzyme is a rapid and monotonic event, which reflects the rate of the proteolytic release of the inhibitory pro-segment and the weaker nature of its interactions with the enzyme moiety compared with procarboxypeptidases of the A1 type, Three main forms of the pro-segment (96, 94, and 92 amino acids), with no inhibitory capability in the severed state, and a single mature carboxypeptidase A2 are produced during this process. No further proteolysis of these pro-segments by the generated carboxypeptidase A2 occurs, in contrast with observations made in other procarboxypeptidases (A1 and B), This differential behavior is a result of the extreme specificity of carboxypeptidase A2.

The transition state for the folding pathway of the activation domain of human procarboxypeptidase A2 (ADA2h) has been analyzed by the protein engineering approach. Recombinant ADA2h is an 81-residue globular domain with no disulfide bridges or cis-prolyl bonds, which follows a two-state folding transition. Its native fold is arranged in two α-helices packing against a four-stranded β-sheet. Application of the protein engineering analysis for 20 single-point mutants spread throughout the whole sequence indicates that the transition state for this molecule is quite compact, possessing some secondary structure and a hydrophobic core in the process of being consolidated. The core (folding nucleus) is made by the packing of α-helix 2 and the two central β-strands. The other two strands, at the edges of the β-sheet, and α-helix 1 seem to be completely unfolded. These results, together with previous analysis of ADA2h with either of its two α-helices stabilized through improved local interactions, suggest that α-helix 1 does not contribute to the folding nucleus, even though it is partially folded in the denatured state under native conditions. On the other hand, α-helix 2 folds partly in the transition state and is part of the folding nucleus. It is suggested that a good strategy to improve folding speed in proteins would be to stabilize the helices that are not folded in the denatured state but are partly present in the transition state. Comparison with other proteins shows that there is no clear relationship between fold and/or size with folding speed and level of structure in the transition state of proteins.

A full-length cDNA clone for preprocarboxypeptidase B from human pancreas has been isolated and sequenced. The open reading frame is 1254 bp in length, encoding a protein of 417 amino acids that includes a leader signal peptide of 15 amino acids and a 95-amino acid-long pro-segment. It contains two differences when compared to the sequence reported for pancreas-specific protein, a human serum marker for acute pancreatitis identified as procarboxypeptidase B. The main difference is a previously unreported Cys at position 138, which is needed for the formation of one of the three disulphide bridges. Sequence alignments between human procarboxypeptidases A1, A2 and B and other known forms show that the most conserved region is the enzyme moiety followed by the globular domain of the pro-segment. The maximum variability is found in the connecting region between moieties. The known three-dimensional structures of procarboxypeptidases from bovine and porcine species have been used to model all three human procarboxypeptidases and also to estimatethe interaction energies between the different parts of the molecules, in an attempt to gain insight into the structural features responsible for the differences observed in the functionality of the proenzymes, particularly in their proteolytic activation pathways. Taken together, the results obtained confirm that the main determinant for the rate and mode of activation of procarboxypeptidases is the strength of the interaction between the enzyme and the globular domain of the pro-segment, the connecting segment playing a complementary role.

Background: Extensive studies of peptide conformation have provided reasonable knowledge of the rules determining helix stability. This knowledge can be used to stabilize proteins against chemical and thermal denaturation. This has been done in two proteins: the chemotactic protein from Escherichia coli, Che Y (a 129 aa alpha/beta parallel protein with five alpha-helices, which shows an accumulating intermediate during refolding) and the activation domain of human procarboxypeptidase A2, ADA2h (a 81 aa alpha+beta protein domain, with two alpha-helices, which follows a two-state mechanism). As the introduced stabilizing interactions are local in nature, the energy balance between the contribution of local and nonlocal interactions changes considerably. Recent theoretical analyses of protein folding using simplified models have indicated that optimization of folding speed requires this balance to be biased towards nonlocal interactions. To determine whether this is the case, we study here the folding kinetics of two ADA2h mutants in which alpha-helix 1 (mutant M1) or 2 (mutant M2) has been stabilized through local interactions, as well as the equilibrium and kinetic behaviour of a double mutant (DM) in which both helices have been stabilized.Results: The stability of DM is considerably enhanced with respect to wild type (WT) and this mutant can be considered as a thermoresistant protein (T-m > 363 K). The thermodynamic parameters obtained by chemical denaturation (urea and GdnHCl) show that DM is similar to 2.6 kcal mol(-1) more stable than WT. The effects on folding kinetics are different in each of the single mutants. MI shows very little effect in refolding, while its unfolding is greatly decelerated with respect to WT. M2 shows, together with a deceleration in unfolding, a significant acceleration in refolding. As with equilibrium parameters, the kinetics of the double mutant can be explained by the simple addition of the effects found in each single mutant. Interestingly enough, the refolding slope m(kf) in mutants M2 and DM is smaller than in the wild-type and M1 mutant.Conclusions: Thermoresistance can be achieved, in some cases, by increasing favourable native local interactions. The balance between local and nonlocal interactions can be significantly changed in some proteins and still keep a cooperative unfolding transition similar to that of the wild type. The introduction of favourable local interactions by mutational redesign can also be used to increase the folding speed of certain proteins, showing that not all proteins in nature have been optimized for rapid folding, contrary to what has been theoretically indicated. This behaviour is probably also shared by other polypeptides with highly unstructured denatured states. All these phenomena have been shown experimentally in ADA2h by mutations that increase helix stability. However, the effects promoted for such an approach in proteins with residual structure and/or intermediates in the denatured ensemble could be different. This has been shown by experiments performed on CheY in which the cooperativity of the folding process was greatly affected.

Background: Increasing protein stability is a major goal of protein engineering because of its potential industrial and pharmacological applications, Several different rule-of-thumb strategies have been employed for such a purpose but a general rational method is still lacking, Recently, there has been significant progress in our understanding of the interactions responsible for helix stability in monomeric peptides and this information has been included in algorithms based on the helix/coil transition theory, We set out to investigate whether it is possible to use these algorithms to rationally increase protein stability,Results: Using a helix/coil transition algorithm, AGADIRms, we have designed mutations affecting solvent exposed residues which, as predicted, significantly increase the helical stability in aqueous solution of peptides corresponding to the two a-helices of the activation domain of procarboxipeptidase A, Introduction of the same mutations in the protein results in proteins more resistant to urea or temperature denaturation, and there is a qualitative agreement between the expected and observed increases in stability,Conclusions: In this work we demonstrate that by using a helix/coil algorithm to design helix-stabilizing mutations on the solvent-exposed face of helices, it is possible to rationally increase the stability of proteins. (C) Current Biology Ltd

The activation domain of human procarboxypeptidase A2 (ADA2h), a globular open-sandwich alpha + beta domain with 80 residues and no disulfide bridges, has been studied by thermodynamic and kinetic analysis. Equilibrium denaturation by urea or temperature is fully reversible at pH 7.0 and fits to a two-state transition. The Gibbs energy of unfolding extrapolated to null concentration of chemical denaturant, delta GH2O, at pH 7.0 and 298 K, is calculated to be 17.0 +/- 1 kJ mol-1, which is within experimental error of the value determined by differential scanning calorimetry, 15.1 +/- 2 kJ mol-1. Kinetics of unfolding and refolding followed by fluorescence do not show the presence of any kinetic intermediate accumulating in the folding reaction. A value for delta GH2O of 17.9 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data. All these data indicate that the folding pathway of this domain is consistent with a two-state model (with the exception of the cis-Pro intermediates). More importantly, the analysis of this and several other small domains or proteins supports the hypothesis that stable kinetic folding intermediates are not necessary for a protein to fold. There seems to be a relationship between the size of a protein and the presence of stable kinetic intermediates. Globular proteins with less than 80 residues and no disulfide bonds follow a two-state transition, while proteins larger than 100 residues present stable kinetic folding intermediates.

The activation process of porcine pancreatic procarboxypeptidase B (pro-CPB) has been studied in detail by a number of complementary methodologies, and a description of the molecular events that lead to the generation of active carboxypeptidase B (CPB) has been deduced. The generated CPB participates in the degradation of its own activation segment by excising C-terminal residues from fragments produced by tryptic proteolysis. The trimming action of CPB is, however, not essential for the release of a fully functional enzyme, in contrast to what was previously reported for porcine procarboxypeptidase A (pro-CPA). In the model presented here, the activation process is solely dependent on the first tryptic cleavage, at the limit between the activation segment and the enzyme region, and the former piece loses all of its inhibitory capacity once severed from the proenzyme. The use of heterologous inhibitors of CPB activity during the study of the tryptic activation process of pro-CPB has been required for the capture of short-lived, otherwise nondetectable, intermediates. This has allowed a complete description of the process and shown that the first proteolytic action of trypsin can also take place on a second target bond. Structural considerations that take into account the three-dimensional structures of the A and B forms of the proenzymes lead us to propose that the differences in conformation at the region that connects the globular activation domain to the enzyme are the main responsible elements for the differences observed in the activation processes of both proenzymes.