Protein and Peptide Letters - Volume 12, Issue 3, 2005

The three-dimensional structure of a protein depends on its amino acid sequence, as do the stability and the folding mechanism. The first milestones in the study of protein folding took place at the beginning of the 20th century: in 1911, Chick and Martin (J. Physiol. 43:1) showed that proteins can be denaturated; in 1931, Wu (Chin. Physiol. 1:219) showed that the process of denaturation involves unfolding; and in 1931, Anson and Mirsky (Phys. Chem. 35:185) showed that the process of unfolding can be reverted. Almost a century later, the convergence of theoretical and experimental work has started to unify the current view of the folding process. This special issue on Protein Folding & Misfolding deals with the biological and physical aspects of this important process. The leading review in this issue, written by Ramos and Ferreira, gives a general view of the field throughout the description of the intermediates, the transition states, the models on protein folding, the biotechnological challenges caused by protein misfolding and aggregation, and the dreadful consequences that misfolding and aggregation can have on conformational diseases. The review by Pereira de Araújo, centered on the use of minimalist models to understand the thermodynamics of cooperative protein folding, addresses the importance of the use of computational methods to understand protein folding. Jamin gives an overview on the folding process of one of the best studied models for protein folding, apomyoglobin, with special attention to the characterization of its folding kinetics. The reviews by Spyracopolous, on the use of nuclear magnetic resonance spectroscopy to determine thermodynamic parameters of proteins, and by Correa and Farah, on the use of 5-hydroxytryptophan as a fluorescence probe to monitor structural properties of proteins, emphasize the importance of spectroscopic techniques as tools for the investigation of folding and stability. Differently from well-behaved proteins that are usually stable at several different conditions, some proteins have a strong tendency to aggregate or to form fibrils, which have medical relevance because these proteins are associated with conformational diseases. In her review, Foguel neatly presents the interpretation of current results in the formation of fibrils by transthyretin using high hydrostatic pressure to investigate the aggregate states of this protein. Cordeiro and Silva center their review on the possible causes of the conversion of the prion protein into its infectious form, pointing out the nucleotide interactions as active components in this reaction. Although we find protein folding very complex, it takes place continually inside cells, partly because of the action of chaperones, proteins involved in a complex machinery in keeping other proteins in their folded states. Chaperones are the theme of the review by Borges and Ramos that summarizes the recent findings in this field. Throughout the articles in this special issue, we see that the final picture emerging from protein folding studies is that this process is governed by simplistic principles. While stability depends more on specific inter-atomic contacts between amino acid residues, the mechanism of folding appears to depend more on the global geometry, or topology, of the native structure. The determination of the protein folding principles is opening a new era in which the structural prediction and design of novel protein structures from the corresponding amino acid sequences are becoming possible. However, considerable work still needs to be done so that we can fully understand how the amino acid sequence encodes the characteristics related to the shape of the folding funnel and to the overall topology of the native protein.

Proteins carry out many vital cellular functions determined by their precise 3-dimensional structures (the native conformations). Understanding how proteins fold has long been a major goal and can be of great therapeutic value. Failure to reach or maintain the correct folded structure can have serious consequences, as in the conformational diseases. The ultimate goal of folding studies is to predict structure from sequence, allowing the design of new functional proteins and prevention of aberrant disease-associated conformations.

Basic concepts about two-state, cooperative protein folding and its relation to first-order phase transitions are reviewed. Minimalist models capable of reproducing the required free energy barrier between folded and unfolded macroscopic states are described. A significantly more restrictive “calorimetric” criterion is also discussed, which is based on direct comparison between model and experimental heat capacities with additional assumptions about conformational enthalpy variation in the unfolded state.

Apomyoglobin (apoMb) folds through at least two partially folded forms that are detected both as transient intermediates during folding/unfolding kinetics or as stable intermediates at equilibrium. Here, I summarize the results of recent kinetic studies, which combined with detailed characterizations of equilibrium forms of the protein, provide a very detailed picture of apoMb folding process. The data are consistent with a linear U Ia Ib N model where compaction and structure are progressively acquired.

Protein dynamics and thermodynamics can be characterized through measurements of relaxation rates of side chain 2H and 13C, and backbone 15N nuclei using NMR spectroscopy. The rates reflect protein motions on timescales from picoseconds to milliseconds. Backbone and methyl side chain NMR relaxation measurements for several proteins are beginning to reveal the role of protein dynamics in protein stability and ligand binding.

5-Hydroxytyptophan is a fluorescent tryptophan analog that can be incorporated into recombinant proteins expressed in E. coli and is particularly useful in studies of biological systems that involve supermolecular aggregates of more than one protein. Here we review the varied applications of 5-hydroxytryptophan to study structure, interactions, conformational change and dynamics in complex protein systems.

High hydrostatic pressure (HHP) is a powerful tool to study protein folding and the dynamics and structure of folding intermediates. Aggregates and amyloids, derived from partially folding intermediates at the junction between productive and off-pathway folding, have been studied as well, which promises better understanding of the protein misfolding diseases. Here is summarized the recent data we have collected with transthyretin under pressure.

The main hypothesis for prion diseases proposes that the cellular protein (PrPC) can be altered into a misfolded, β-sheet-rich isoform (PrPSc). We describe here that host nucleic acid may catalyze the conversion between PrPC and PrPSc isoforms, by reducing the protein mobility and by making the protein-protein interactions more likely. We summarize the findings, focusing in the biological relevance of the catalytic action of nucleic acid.

Molecular chaperones are one of the most important cell defense mechanisms against protein aggregation and misfolding. These specialized proteins bind non-native states of other proteins and assist them in reaching a correctly folded and functional conformation. Chaperones also participate in protein translocation by membranes, in the stabilization of unstable protein conformers and regulatory factors, in the delivery of substrates for proteolysis and in the recovery of proteins from aggregates.

Calcitonin (CT), a peptide hormone that is widely used for the treatment of osteoporosis, Paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients, is produced by the para-follicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Fish calcitonin, like eel calcitonin (eCT), is more potent and longer lasting than human CT and is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated eCT in recombinant Streptomyces avermitilis. A phylogenetic analysis was performed with all the known CT amino acid sequences.

Copper was added to truncated, recombinant cystathionine β-synthase (CBS), and the enzyme activity was assessed by measuring the production of cystathionine. 10microM copper significantly decreased CBS activity by 50% while 25microM copper decreased CBS activity by 70%. This inhibition was negated when an analog of the N-terminus of human albumin, Asp-Ala-His-Lys (DAHK), a strong transition metal binding peptide, was added. The use of copper chelators could significantly reduce in vivo homocysteine levels.

Peptide analogues of Tendamistat which include the most essential residues linked by novel w-amino acids (X,Y,Z: H2N-(CH2)n-CO2H, where n=2-10) were designed, synthesized (Ac-Tyr15-X-Trp18-Arg19-Tyr20-Y-Thr55-Z-Asp58- Gly59-Tyr60-Ile61-Gly62-NH2), and analyzed for α-amylase inhibitory activity. Native dipeptide spacers sometimes were left intact at X and Z. Analogues demonstrated competitive inhibition with Ki values ranging from 23 to 767 μM. 8- Aminooctanoic acid was the optimal linker at Y, while longer linkers were favored at the other positions.

A practical method for the synthesis of an important precursor of constrained peptides involving only three steps from commercial inexpensive precursors is reported.

The mutation, conferring streptomycin and deoxyglucose resistance on cells, had profound effect on the kinetic and thermodynamic parameters inferring thermostabilization of ß-glucosidase from mutant 51 SMr of Cellulomonas biazotea. Free energy of activation for substrate binding, enthalpy and entropy of activation for irreversible denaturation of mutant-derived enzyme were decreased compared with enzyme from wild organism suggesting that the mutation partly stabilized the enzyme and that mutation made it more reactive.

To investigate the role of arginine in the folding of D-aminoacylase, seven arginine residues, R26, R152, R296, R302, R354, R377, and R391, among twelve arginine residues highly conserved in D-aminoacylase, N-acyl-D-aspartate amidohydrolase (D-AAase), and N-acyl-D-glutamate amidohydrolase (D-AGase) from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were substituted with lysine by site-directed mutagenesis. The mutants, R26K, R152K, R296K, and R302K were identified as mutations that increase partitioning of the enzyme into inclusion bodies. No mutants with substitutions within the carboxyterminal segment were found to increase partitioning into inclusion bodies (R354K, R377K, and R392K). These results suggest that arginine residues that position between the N-terminus and central region can play an important role in facilitating folding or stabilizing the structure of D-aminoacylase. By anaerobic cultivation, the production level of R302K in the soluble fraction was improved. Coexpression of the DnaKDnaJ- GrpE chaperone assisted the folding of R302K, and reduced the effect of the aeration conditions on the solubility of R302K. We hypothesized that R302K requires a larger amount of chaperones for efficient folding than the wild type enzyme.

In yeast two-hybrid system, rat 12-lipoxygenase (12-LO) bound to complete (390 amino acids) or the Nterminus truncated form of human p47 phox, but not to the C-terminus truncated form (residues 1-286). When glutathione S-transferase fused human p47phox was added to an in vitro 12-LO enzyme activity assay, formation of 12- hydroperoxyeicosatetraenoic acid was reduced significantly compared to the C-terminus truncated form. These results indicate that C-terminus of p47phox is important for its interaction to rat 12-LO.

We have purified and characterized pig and bovine milk lactadherins. Studies by circular dichroism spectroscopy indicate that the two proteins present a similar folding pattern. Results have been discussed in terms of their affinity for pig zona pellucida in order to use these proteins as analogs of pig sperm lactadherin in gamete studies.