Clitocypin, a new cysteine proteinase inhibitor, is monomeric: impact on the mechanism of folding

The molecular mass of clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, has been determined by analytical ultracentrifugation and gel exclusion chromatography. The result is in agreement with the formula mass of 16.8 kDa, demonstrating that the inhibitor is a monomer in aqueous solution. This enables the kinetics of unfolding and refolding to be interpreted in terms of folding in a kinetically two state, highly cooperative transition from the thermally unfolded state.     

Conformational stability and folding mechanisms of dimeric proteins

The folding of multisubunit proteins is of tremendous biological significance since the large majority of proteins exist as protein-protein complexes. Extensive experimental and computational studies have provided fundamental insights into the principles of folding of small monomeric proteins. Recently, important advances have been made in extending folding studies to multisubunit proteins, in particular homodimeric proteins. This review summarizes the equilibrium and kinetic theory and models underlying the quantitative analysis of dimeric protein folding using chemical denaturation, as well as the experimental results that have been obtained. Although various principles identified for monomer folding also apply to the folding of dimeric proteins, the effects of subunit association can manifest in complex ways, and are frequently overlooked. Changes in molecularity typically give rise to very different overall folding behaviour than is observed for monomeric proteins. The results obtained for dimers have provided key insights pertinent to understanding biological assembly and regulation of multisubunit proteins. These advances have set the stage for future advances in folding involving protein-protein interactions for natural multisubunit proteins and unnatural assemblies involved in disease.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Unfolding energetics and conformational stability of DLC8 monomer

To understand the rules governing the protein folding process it is essential to study the stability and unfolding of small monomeric proteins. Here, I present the pH dependent thermal unfolding energetics and conformational stability analysis of monomeric Dynein light chain protein (DLC8) in the pH range 3.5-2.0. DLC8 is the smallest and the most conserved light chain among the light chains of the dynein motor assembly. Thermal unfolding of DLC8 monomer is much complex with the presence of transient intermediates, which is in contrast to the notion that small proteins unfold via simple two-state process. The unfolding seems to be more cooperative at lower pH and the temperature of highest conformational stability (T(s)) is found to be maximum (295.7 K) at pH 2.76. Stability curves have been simulated to understand the thermodynamic parameters that govern the shapes of the experimentally obtained curves. Further, an effort has been made to correlate the observed differences in the denaturation energetics with the protein sequence in order to throw light on the structure-folding paradigm of the DLC8 monomer.     

Analysis of the stability and function of nucleoplasmin through cysteine mutants

Xenopus laevis nucleoplasmin is a pentameric nuclear chaperone. The relation between the structure and the multifunctional aspects of the molecule has not yet been clearly established. In the course of analysing a C-terminally His-tagged recombinant version of the region equivalent to the trypsin resistant core (r-NP142) of the molecule, we found that this domain exhibited a substantially decreased oligomerization potential. To better understand the role of the three cysteines of nucleoplasmin on its pentameric functional structure, we have selectively mutated these residues to serine and generated three mutants (C15S, C35S, and C45S) both for the complete recombinant nucleoplasmin (r-NP) and the truncated r-NP142 non-tagged forms. We demonstrate that there are no disulphide bridges stabilizing either the monomer or the pentamer. Neither C15S nor C35S has any structural effects, while the mutation C45S abolishes the ability of r-NP142 to pentamerize. This structural impairment suggests that hydrophobic interactions of Cys 45 are critical for the stability of the protein. Our studies allow to analyse for the first time the structural and functional properties of nucleoplasmin in its monomeric form.     

Reinvestigating the codon and amino acid usage of S. cerevisiae genome: a new insight from protein secondary structure analysis

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance for quite a long time now. Taking advantage of publicly available gene expression data for Saccharomyces cerevisiae, a systematic analysis of the codon and amino acid usages in two different coding regions corresponding to the regular (helix and strand) as well as the irregular (coil) protein secondary structures, have been performed. Our analyses suggest that apart from tRNA abundance, mRNA folding stability is another major evolutionary force in shaping the codon and amino acid usage differences between the highly and lowly expressed genes in S. cerevisiae genome and surprisingly it depends on the coding regions corresponding to the secondary structures of the encoded proteins. This is obviously a new paradigm in understanding the codon usage in S. cerevisiae. Differential amino acid usage between highly and lowly expressed genes in the regions coding for the irregular protein secondary structure in S. cerevisiae is expounded by the stability of the mRNA folded structure. Irrespective of the protein secondary structural type, the highly expressed genes always tend to encode cheaper amino acids in order to reduce the overall biosynthetic cost of production of the corresponding protein. This study supports the hypothesis that the tRNA abundance is a consequence of and not a reason for the biased usage of amino acid between highly and lowly expressed genes.     

Thermodynamics and stability of the PAAD/DAPIN/PYRIN domain of IFI-16

The PAAD domain is a conserved domain recently identified in more than 35 human proteins that are involved in apoptosis and inflammatory signaling pathways. Structural studies have confirmed that this domain belongs to the death domain superfamily which includes PAAD/CARD/DED/DD families. Recently, the 3D structures determined by NMR of NALP1 and ASC PAAD domain, members of the PAAD family, have shown that it is composed of a 6 helix bundle as with other death domain family members. However, helix-3 in the solved structures is unordered in solution. In this study we compare the thermodynamic, folding and stability properties of different members of the PAAD and CARD families and investigate structural conformational changes induced by the helix inducers trifluoroethanol and SDS on the PAAD domain of IFI16 and on the CARD domain of RAIDD. We show that inside the PAAD and CARD families, members have similar thermodynamic properties, however, the DeltaG of folding for PAAD and CARD members are, respectively, -1.4 and -5.5 kcal mol(-1). This difference is attributed to less alpha helical content for PAAD due to the unfolding of helix-3 that lowers bonded energy and increases disorder when compared to CARD members. Despite identical fold between PAAD and CARD families but limited sequence identity, there are striking differences in the thermodynamics of both families.     

Circular dichroism and laser Raman spectroscopic analysis of the secondary structure ofCerebratulus lacteus toxin B-IV

The secondary structure ofCerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical -helical peaks at 191, 208, and 222 nm. At neutralpH, the -helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% -helix estimate. Both CD and Raman spectroscopic methods failed to detect any -sheet structure. The spectroscopic analyses revealed significantly more -helix and less -sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49–55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing thatCerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at highpH are consistent with this interpretation.     

Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor fromSorghum bicolor seedlings

A 711-bp cDNA encoding a cysteine proteinase inhibitor (cystatin) was isolated from a cDNA library prepared from 7–10 cmSorghum bicolor seedlings. The nearly full-length cDNA clone encodes 130 amino acid residues, which include the Gln-Val-Val-Ala-Gly motif, conserved among most of the known cystatins as a probable binding site for cysteine proteinases. The amino acid sequence of sorghum cystatin deduced from the cDNA clone shows significantly homology to those of other plant cystatins. The sorghum cystatin expressed inE. coli showed a strong papain-inhibitory activity.     

Circular dichroism study on the secondary structural change induced by complex formation of a peptide derived from a CD4 binding site of HIV-1 envelope glycoprotein gp120 and a peptide from the N-terminal domain of CD4

Summary Circular dichroism spectroscopy was used to study the conformational change of a peptide containing a CD4 binding region of HIV-1 envelope glycoprotein gp120 complexed with a CD4 fragment. In free solution the gp120 peptide exists primarily as -sheet and random coil. Upon association with the peptide, encompassing a critical gp120 binding site on the extracellular domain 1 of CD4, the -helical content of the complex relative to that of the two component peptides increases significantly, at the expense of random coil and turn. An increase in the helix structure for the gp120 peptide, but not the CD4 peptide, was observed in 30% trifluoroethanol (TFE)/H₂O (v:v) solution. The conformational change in the gp120 C4 peptide when complexing with CD4 is proposed as part of the process that facilitates the membrane fusion between the virion and its target cell.Abbreviations CD circular dichroism - HIV human immunodeficiency virus - AIDS acquired immunodeficiency syndrome - Fmoc 9-fluorenylmethoxycarbonyl - mAb monoclonal antibody - gp glycoprotein - TFE trifluoroethanol - HPLC high-performance liquid chromatography     

Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses

Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically‐defined spectroscopic signatures derived from proteins with known three‐dimensional structures. Many proteins identified as being “Intrinsically Disordered Proteins” have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as “other”, “random coil”, “unordered”, or “disordered”. However the “other” category can also include polyproline II (PPII)‐type structures, whose spectral properties have not been well‐distinguished from those of unordered structures. In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII‐type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid‐containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins.     

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: Protein stability and denatured state residual structure

Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.     

Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate

Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.     

CSSI-PRO: a method for secondary structure type editing,assignment and estimation in proteins using linear combination of backbone chemical shifts

Estimation of secondary structure in polypeptides is important for studying their structure, folding and dynamics. In NMR spectroscopy, such information is generally obtained after sequence specific resonance assignments are completed. We present here a new methodology for assignment of secondary structure type to spin systems in proteins directly from NMR spectra, without prior knowledge of resonance assignments. The methodology, named Combination of Shifts for Secondary Structure Identification in Proteins (CSSI-PRO), involves detection of specific linear combination of backbone ¹H^α and ¹³C′ chemical shifts in a two-dimensional (2D) NMR experiment based on G-matrix Fourier transform (GFT) NMR spectroscopy. Such linear combinations of shifts facilitate editing of residues belonging to α-helical/β-strand regions into distinct spectral regions nearly independent of the amino acid type, thereby allowing the estimation of overall secondary structure content of the protein. Comparison of the predicted secondary structure content with those estimated based on their respective 3D structures and/or the method of Chemical Shift Index for 237 proteins gives a correlation of more than 90% and an overall rmsd of 7.0%, which is comparable to other biophysical techniques used for structural characterization of proteins. Taken together, this methodology has a wide range of applications in NMR spectroscopy such as rapid protein structure determination, monitoring conformational changes in protein-folding/ligand-binding studies and automated resonance assignment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of a Chinese cabbage cysteine proteinase inhibitor, BrCYS1, retards seed germination and plant growth in transgenic Tobacco plant

Phytocystatins are plant cysteine proteinase inhibitors that regulate endogenous and heterologous cysteine proteinases of the papain family. A cDNA encoding the phytocystatin BrCYS1 (Brassica rapa cysteine proteinase inhibitor 1 ) has been isolated from Chinese cabbage (B. rapa subsp.pekinensis) flower buds. In order to explore the role of this inhibitory enzyme, tobacco plants (Nicotiana tabacum L. cv. Samson) containing altered amounts of phytocystatin were generated by over-expressingBrCYS1 cDNA in either the sense or the antisense configuration. The resulting plants hadin vitro enzyme inhibitory activities that were over 10% of those detected in wild type plants. The transgenic plants exhibited retarded seed germination and seedling growth and a reduced seed yield, whereas these properties were enhanced in antisense plants. These data suggest that BrCYS1 participates in the control of seed germination, post-germination and plant growth by regulating cysteine peptidase activity.     

Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.     

Structure and thermal stability of the extracellular fragment of human transferrin receptor at extracellular and endosomal pH

Fourier transform infrared spectroscopy has been used to study the solution structure and thermal stability of the extracellular fragment of human transferrin receptor (tfRt) at extracellular and endosomal pH. At extracellular pH tfRt is composed of 56% -helix, 19% β-sheet and 14% turns. Upon acidification to endosomal pH the -helical content of the protein is reduced and the β-sheet content increased by nearly 10%. At extracellular pH, the midpoint temperature of thermal denaturation (T_m) for the loss of secondary and tertiary structure, and the formation of aggregated structures, is 71°C. At endosomal pH this temperature is reduced by ≈ 15°C. The apparent entropies of thermal denaturation indicate that the native structure of tfRt at endosomal pH is far more flexible than at extracellular pH.     

A major cysteine proteinase, EPB, in germinating barley seeds: structure of two intronless genes and regulation of expression

The barley cysteine proteinase B (EPB) is the main protease responsible for the degradation of endosperm storage proteins providing nitrogenous nutrients to support the growth of young seedlings. The expression of this enzyme is induced in the germinating seeds by the phytohormone, gibberellin, and suppressed by another phytohormone, abscisic acid. In situ hybridization experiments indicate that EPB is expressed in the scutellar epithelium within 24 h of seed germination, but the aleurone tissue surrounding the starchy endosperm eventually becomes the main tissue expressing this enzyme. The EPB gene family of barley consists of two very similar genes, EPB1 and EPB2, both of which have been mapped to chromosome 3. The sequences of EPB1 and EPB2 match with the two previously published cDNA clones indicating that both genes are expressed. Interestingly, neither of these genes contain any introns, a rare phenomenon in which all members of a small gene family are active intronless genes. Sequence comparison indicates that the barley EPB family can be classified as cathepsin L-like endopeptidases and is most closely related to two legume cysteine proteinases (Phaseolus vulgaris EP-C1 and Vigna mungo SHEP) which are also involved in seed storage protein degradation. The promoters of EPB1 and EPB2 have been linked to the coding sequence of a reporter gene, GUS, encoding -glucuronidase, and introduced into barley aleurone cells using the particle bombardment method. Transient expression studies indicate that EPB promoters are sufficient to confer the hormonal regulation of these genes.     

Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean)

Herein, we report the purification and biochemical characterization of a novel bi-functional protein proteinase/amylase inhibitor from the dietary leguminous pulse Phaseolus aureus Roxb. (Vigna radiata L.) by means of acetic acid precipitation, salt fractionation, ion-exchange chromatography (DEAE-cellulose) and affinity chromatography on trypsin-sepharose column. P. aureus inhibitor is a bi-functional inhibitor since it exhibits inhibitory activity towards trypsin-like and alpha-chymotrypsin-like serine proteinases as well as against alpha-amylases. It is a helix-rich protein (Mr 13,600) containing approximately eight tyrosines, one tryptophan and two cystines. N-terminal sequence alignment reveals no homology to other proteinase inhibitors reported from Phaseolus sp. thereby confirming that it is a novel inhibitor. Inhibitory activity measurements show that the inhibitor is quite stable even at extremely high temperatures and is only slightly affected by pH changes. Circular dichroism (CD) conformational studies revealed some changes in its near- as well as far-ultraviolet spectrum at extremes of pH and temperature. Treatments with trypsin for varying time periods did not alter its proteolytic inhibitory activity but caused some reduction in its amylase inhibitory activity.