Lessons on protein structure from interleukin-4: All disulfides are not created equal.

Protein phosphorylation represents a ubiquitous control mechanism in living cells. The structural prerequisites and consequences of this important post-translational modification, however, are poorly understood. Oncoprotein 18/stathmin (Op18) is a globally disordered phosphoprotein that is involved in the regulation of the microtubule (MT) filament system. Here we document that phosphorylation of Ser63, which is located within a helix initiation site in Op18, disrupts the transiently formed amphipathic helix. The phosphoryl group reduces tubulin binding 10-fold and suppresses the MT polymerization inhibition activity of Op18's C-terminal domain. Op18 represents an example where phosphorylation occurs within a regular secondary structural element. Together, our findings have implications for the prediction of phosphorylation sites and give insights into the molecular behavior of a globally disordered protein.

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.

Using nuclear magnetic resonance spectroscopy to study molten globule states of proteins

Molten globule-state protein structure: Perspectives from food processing applications

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

The Molten Globule of β2-Microglobulin Accumulated at pH 4 and Its Role in Protein Folding

Physicochemical and structural properties of lunasin revealed by spectroscopic, chromatographic and molecular dynamics approaches

DsbA is a protein found in the periplasm of Escherichia coli that is required for the formation of disulfide bonds in secreted proteins. It contains only two cysteine residues, which can form reversibly a very unstable disulfide bond that has been proposed to be the oxidant that introduces disulfide bonds into secreted proteins. The present study investigates the effect of DsbA on the well-characterized disulfide-coupled refolding processes of BPTI and of alpha-lactalbumin. Disulfide-bonded DsbA in stoichiometric amounts proved to be a very potent donor of disulfide bonds to reduced BPTI but showed little catalytic activity at neutral pH in the presence of a glutathione redox buffer. In contrast to the related eukaryotic enzyme protein disulfide isomerase, DsbA did not substantially catalyze the usual intramolecular disulfide bond rearrangements of quasi-native folding intermediates of BPTI. Neither did DsbA catalyze the intramolecular rearrangements observed in the three disulfide-bonded "molten globule" form of alpha-lactalbumin at neutral pH. Thiol-disulfide exchange is normally very slow at acidic pH but occurs rapidly with DsbA; consequently, DsbA catalyzed the disulfide folding of BPTI under acidic conditions. It was then possible to detect some increase in the rates of disulfide rearrangements, but only with stoichiometric amounts of DsbA and on the hour time scale. These results suggest that the primary role of DsbA in the bacterial periplasm is to introduce disulfide bonds into newly secreted proteins.

Effect of n-alkyl trimethylammonium bromides on folding and stability of alkaline and acid-denatured cytochrome c: A spectroscopic approach

Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.

The study of the mechanisms underlying formation of molten globule states in proteins is gaining growing interest due to recent discoveries concerning the involvement of this folding intermediate in several biological processes and to the possibility it offers to clarify the general process of protein folding. Different spectroscopic techniques, such as fluorescence, circular dichroism, nuclear magnetic resonance and infrared, have proved to be invaluable tools in the detection and characterization of molten globule states. In this review, the use of Fourier Transform infrared (FT-IR) spectroscopy to shed light on the formation of molten globules is discussed. Due to its ability to simultaneously probe the secondary structure and compactness/flexibility of protein samples prepared in deuterated media, FT-IR spectroscopy is particularly well suited to detect temperature-induced molten globule states in small, β-sheet-rich proteins. Several approaches for analysis and interpretation of infrared spectra are provided, such as second derivatives, Amide I′ position and extent of H/D exchange plots, difference spectra and 2D correlation analysis. Experimental cases are also provided as examples.

In the last decades osteoporosis has become a major subject on the field of drug discovery and design. One of the enzymes recently considered important to use as a target for theses drugs is the enzyme H+-VO-ATPase. This proton pump is located in the osteoclast cells, which are positioned at the bone surface. These enzymes control the proton flux to the bone surface and consequently bone resorption. One major task on drug design is the knowledge of the secondary and tertiary structure of the enzyme under study. The topology of the V-ATPase protein complex has been largely established, however, only the three-dimensional structure of some individual subunits is known up to the time this thesis was printed. The work presented in this thesis focuses on the proton translocation channel located in subunit a of the V-ATPase complex. For this purpose, we designed two peptides, consisting of 25 and 37 amino acid residues, representing the seventh transmembrane segment of subunit a that encompass the proton translocation channel as well as the region of interest for possible inhibitors. Using a combination of NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy we analysed the conformation of these V-ATPase peptides in different membrane-mimicking environments: aqueous solutions of SDS (sodium dodecyl sulphate) and amphipols, and the organic solvent DMSO (dimethylsulphoxide). The conformation of the V-ATPase peptides in SDS micelles was studied by CD spectroscopy, however, due to their low solubility NMR spectroscopy turned out to be impossible. The CD results showed that the size of the peptide can drastically alter the solubilization in SDS. For the 37-residue V-ATPase peptide the overall conformation was a-helical and not dependent on the SDS concentration. On the other hand, the conformation of the 25-residue V-ATPase peptide depended on the peptide to SDS ratio changing between an a-helix and β-sheet conformation. As an alternative solubilising agent for the peptides in aqueous solutions we tested amphipols, a new class of macromolecules that were designed to solubilise transmembrane proteins for NMR and X-ray studies. The CD and tryptophan fluorescence spectroscopy results showed that both peptides aggregated in a β-sheet conformation. The formation of these b‑sheets aggregates might result from the interaction of the arginine residue present in the V-ATPase peptides with the anionic polymer. Such an interaction could prevent the peptide from crossing the hydrophobic core of the particle, preventing the formation of an a‑helix. High-quality high-resolution NMR spectra of the V-ATPase peptides were obtained in DMSO enabling to analyse the atomic structure of the peptides. The use of DMSO on structural studies of transmembrane polypeptides always raised some debate in the literature. This fact motivated us to perform a molecular dynamics study to investigate the solvation of the 25-residue V-ATPase peptide by DMSO. From this work, we show that DMSO can provide both polar and apolar environments to the peptide, making it a good membrane-mimicking organic solvent. The NMR study of the 37-residues peptide enabled us to confirm the a-helical conformation and to predict that the transmembrane spanning region of the seventh transmembrane segment is larger than expected. Instead of 25 transmembrane residues, we propose a transmembrane region of 32 residues. Furthermore, the NMR study of the 25-residue peptide lead us to postulate the existence of a hinge region located near the cytoplasmic end of the channel. It is proposed that the presence of this hinge allows the opening and closing of the proton translocation channel and provides flexibility for the channel to act as a binding pocket for inhibitors. The resulting NMR data sets for the two V-ATPase peptides are deposited in the BioMagResbank (BMRB) under the access numbers 15025 and 6878, and the calculated structure ensemble for the 25-residue peptide is deposited in the PDB databank with entry 2NVJ.

Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins. The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (Fl) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state. Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degreesC) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.

beta-Lactamase from Bacillus licheniformis forms a stable compact intermediate state at low pH and moderate salt concentration (the A state), with properties consistent with a molten globule. A single cysteine residue was introduced into this class A beta-lactamase by site-directed mutagenesis at position 166. A spin label was attached to the thiol of this cysteine residue via a disulfide bond as a probe of the side-chain mobility. The mutant protein and the spin-labeled derivative exhibited similar conformational properties to the wild-type enzyme at acidic pH. The A state induced by chloride or trichloroacetate (TCA) anions was characterized by circular dichroism and esr. The A state at pH 0.5 (0.32 M HCl), or at pH 2 in the presence of 8 mM TCA or 0.4 M Cl-, had comparable amounts of secondary structure to the native state but lacked significant tertiary structure, as judged by the lack of near-UV circular dichroism. Analysis of the esr spectral line widths showed that the mobility of the spin label in the A state was similar to that in the native state and much less mobile than in the unfolded state, indicating significant constraints on the side-chain mobility in this region of the molecule in the A state. The implications of this finding to the structure of the A state are discussed.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.