The B domain of protein A retains residual structures in 6 M guanidinium chloride as revealed by hydrogen/deuterium-exchange NMR spectroscopy.

The characterization of residual structures persistent in unfolded proteins is an important issue in studies of protein folding, because the residual structures present, if any, may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the residual structures of the isolated B domain (BDPA) of staphylococcal protein A in 6 M guanidinium chloride. BDPA is a small three-helix-bundle protein, and until recently its folding/unfolding reaction has been treated as a simple two-state process between the native and the fully unfolded states.

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science.

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science.

Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR.

The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution.

The Molten Globule, and Two-State vs. Non-Two-State Folding of Globular Proteins.

From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding.

Exploring the Folding Mechanism of Small Proteins GB1 and LB1.

The computational atomistic description of the folding reactions of the B1 domains, GB1 and LB1, of protein G and protein L, respectively, is an important challenge in current protein folding studies. Although the two proteins have overall very similar backbone structures (β-hairpin-α-helix-β-hairpin), their apparent folding behaviors observed experimentally were remarkably different. LB1 folds in a two-state manner with the single-exponential kinetics, whereas GB1 folds in a more complex manner with an early stage intermediate that may exist on the folding pathway.

Determination of the structural ensemble of the molten globule state of a protein by computer simulations.

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface.

PFDB: A standardized protein folding database with temperature correction.

We constructed a standardized protein folding kinetics database (PFDB) in which the logarithmic rate constants of all listed proteins are calculated at the standard temperature (25 °C). A temperature correction based on the Eyring-Kramers equation was introduced for proteins whose folding kinetics were originally measured at temperatures other than 25 °C. We verified the temperature correction by comparing the logarithmic rate constants predicted and experimentally observed at 25 °C for 14 different proteins, and the results demonstrated improvement of the quality of the database.

An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior.

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet.

Formation of the chaperonin complex studied by 2D NMR spectroscopy.

We studied the interaction between GroES and a single-ring mutant (SR1) of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C) in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]).

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop.

Dissection of the ATP-dependent conformational change cycle of a group II chaperonin.

Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle.

The catalytic domain of topological knot tRNA methyltransferase (TrmH) discriminates between substrate tRNA and nonsubstrate tRNA via an induced-fit process.

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity.

ATP dependent rotational motion of group II chaperonin observed by X-ray single molecule tracking.

Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle.

NMR characterization of the interaction of GroEL with amyloid β as a model ligand.

Here we report an NMR study on the substrate interaction modes of GroEL using amyloid β (Aβ) as a model ligand. We found that GroEL could suppress Aβ(1-40) amyloid formation by interacting with its two hydrophobic segments Leu17-Ala21 and Ala30-Val36, which involve key residues in fibril formation. The binding site of Aβ(1-40) was mapped on a pair of α-helices located in the GroEL apical domain.

The H/D-exchange kinetics of the Escherichia coli co-chaperonin GroES studied by 2D NMR and DMSO-quenched exchange methods.

We studied hydrogen/deuterium-exchange reactions of peptide amide protons of GroES using two different techniques: (1) two-dimensional (1)H-(15)N transverse-optimized NMR spectroscopy and (2) the dimethylsulfoxide-quenched hydrogen-exchange method combined with conventional (1)H-(15)N heteronuclear single quantum coherence spectroscopy. By using these techniques together with direct heteronuclear single quantum coherence experiments, we quantitatively evaluated the exchange rates for 33 out of the 94 peptide amide protons of GroES and their protection factors, and for the remaining 61 residues, we obtained the lower limits of the exchange rates. The protection factors of the most highly protected amide protons were on the order of 10(6)-10(7), and the values were comparable in magnitude to those observed in typical small globular proteins, but the number of the highly protected amide protons with a protection factor larger than 10(6) was only 10, significantly smaller than the numbers reported for the small globular proteins, indicating that significant portions of free heptameric GroES are flexible and natively unfolded.

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state.

Nucleotide-induced conformational changes of tetradecameric GroEL mapped by H/D exchange monitored by FT-ICR mass spectrometry.

Here we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to access E. coli chaperonin GroEL conformation. The ~800 kDa tetradecameric GroEL plays an essential role in the proper folding of many proteins.

The use of spin desalting columns in DMSO-quenched H/D-exchange NMR experiments.

Dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange is a powerful method to characterize the H/D-exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non-protected fast-exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO-quenched H/D-exchange studies of proteins so far reported, lyophilization was used to remove D2 O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two-dimensional nuclear magnetic resonance (2D NMR) spectra.

Structural insights into the stability perturbations induced by N-terminal variation in human and goat α-lactalbumin.

Addition of an extra methionine at the N-terminus by recombinant expression of α-lactalbumin in Escherichia coli significantly destabilizes the protein, and this destabilization has hampered mutational analyses such as the mutational phi-value analysis of the protein. Deletion of residue 1 from the recombinant form recovers the stability in human and goat α-lactalbumin. Here, we thus determined the crystal structures of the residue 1-deletion variants of recombinant human and goat α-lactalbumin, and compared the structures with those of the authentic and recombinant forms.

The molten globule of β(2)-microglobulin accumulated at pH 4 and its role in protein folding.

The acid transition of β(2)-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH4 (25°C). The pH4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state.

Native-state heterogeneity of β(2)-microglobulin as revealed by kinetic folding and real-time NMR experiments.

The kinetic folding of β(2)-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH7.5 to pH2.

Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants.

Equilibria and kinetics of folding/unfolding of α-lactalbumin and its two N-terminal variants were studied by circular dichroism spectroscopy. The two variants were wild-type recombinant and Glu1-deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant α-lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1.

Fibrillogenic propensity of the GroEL apical domain: a Janus-faced minichaperone.

The chaperonin GroEL plays an essential role in promoting protein folding and in protecting against misfolding and aggregation in the cellular environment. In this study, we report that both GroEL and its isolated apical domain form amyloid-like fibrils under physiological conditions, and that the fibrillation of the apical domain is accelerated under acidic conditions. We also found, however, that despite its fibrillation propensity, the apical domain exhibits a pronounced inhibitory effect on the fibril growth of β(2)-microglobulin.

Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.

Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature.