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The reagents and methods for purification and use of the most commonly used denaturants, guanidine hydrochloride (guanidine-HCl) and urea, are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and "oxido-shuffling" (or oxidative regeneration) systems are also described., (Copyright © 2016 John Wiley & Sons, Inc.)

Heat shock proteins (HSP) perform vital cellular functions and modulate cell response pathways to physical and chemical stressors. A key feature of HSP function is the ability to interact with a broad array of protein binding partners as a means to potentiate downstream response pathways or facilitate protein folding. These binding interactions are driven by ATP-dependent conformational rearrangements in HSP proteins. The HSP70 family is evolutionarily conserved and is associated with diabetes and cancer progression and the etiopathogenesis of hepatic, cardiovascular, and neurological disorders in humans. However, functional characterization of human HSP70s has been stymied by difficulties in obtaining large quantities of purified protein. Studies of purified human HSP70 proteins are essential for downstream investigations of protein-protein interactions and in the rational design of novel family-specific therapeutics. Within this work, we present optimized protocols for the heterologous overexpression and purification of either the nucleotide binding domain (NBD) or the nucleotide and substrate binding domains of human HSPA9, HSPA8, and HSPA5 in either Escherichia coli or Saccharomyces cerevisiae. We also include initial biophysical characterization of HSPA9 and HSPA8. This work provides the basis for future biochemical studies of human HSP70 protein function and structure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Trichloroacetic acid (TCA) precipitation of proteins is commonly used to concentrate protein samples or remove contaminants, including salts and detergents, prior to downstream applications such as SDS-PAGE or 2D-gels. TCA precipitation denatures the protein, so it should not be used if the protein must remain in its folded state (e.g., if you want to measure a biochemical activity of the protein)., (© 2014 Elsevier Inc. All rights reserved.)

Backbone-cyclized proteins, with their characteristic stability toward denaturants such as heat and chemicals, are becoming increasingly significant in many applications. Intein-mediated protein cyclization is the most efficient and frequently used method of choice and has been successfully applied to various targets, achieving stable proteins. However, the detection and isolation of the cyclic protein from the linear one after cyclization is very difficult because the backbone-cyclized protein and the linear one (a by-product formed during the cyclization reaction), which originated from the same molecule, are almost identical in terms of their size. Thus, we first developed a split c-myc tag system; the active c-myc tag was formed only in the backbone-cyclized protein and not in the linear by-product from the inactive precursor, and this helps both the detection and purification of the backbone-cyclized proteins. This tag system, which we called a cyclization tag, was further engineered in its sequence to develop an engineered c-myc (e-myc) tag with enhanced efficiency in the backbone cyclization reaction while keeping its specificity toward the commercial antibody intact. Using two different proteins as models, we show that the cyclization tag developed here can be used as a specific tag for the backbone-cyclized protein, thereby facilitating detection and purification., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recently, the role of force in cellular processes has become more evident, and now with advances in force spectroscopy, the response of proteins to force can be directly studied. Such studies have found that native proteins are brittle, and thus not very deformable. Here, we examine the mechanical properties of a class of intermediates referred to as the molten globule state. Using optical trap force spectroscopy, we investigated the response to force of the native and molten globule states of apomyoglobin along different pulling axes. Unlike natively folded proteins, the molten globule state of apomyoglobin is compliant (large distance to the transition state); this large compliance means that the molten globule is more deformable and the unfolding rate is more sensitive to force (the application of force or tension will have a more dramatic effect on the unfolding rate). Our studies suggest that these are general properties of molten globules and could have important implications for mechanical processes in the cell.

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = K(agg)(T-T(0))(2), where K(agg) is the parameter characterizing the initial rate of aggregation and T(0) is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T(0) corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets.

The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain integral membrane protein central to epithelial fluid secretion (see Chapter 21). Its activity is defective in the recessive genetic disease cystic fibrosis (CF). The most common CF-causing mutation is F508del in the first nucleotide binding domain (NBD1) of CFTR. This mutation is found on at least one allele of more than 90% of all CF patients. It is known to interfere with the trafficking/maturation of CFTR through the secretory pathway, leading to a loss-of-function at the plasma membrane. Notably, correction of the trafficking defect by addition of intragenic second-site suppressor mutations, or the alteration of bulk solvent conditions, such as by reducing the temperature or adding osmolytes, leads to appearance of functional channels at the membrane--thus, the rescued F508del-CFTR retains measurable function. High-resolution structural models of NBD1 from X-ray crystallographic data indicate that F508 is exposed on the surface of the domain in a position predicted by homologous ABC transporter structures to lie at the interface with the intracellular loops (ICLs) connecting the transmembrane spans. Determining the relative impact of the F508del mutation directly on NBD1 folding or on steps of domain assembly or both domain folding and assembly requires methods for evaluating the structure and stability of the isolated domain.

Stress, molecular crowding and mutations may jeopardize the native folding of proteins. Misfolded and aggregated proteins not only loose their biological activity, but may also disturb protein homeostasis, damage membranes and induce apoptosis. Here, we review the role of molecular chaperones as a network of cellular defenses against the formation of cytotoxic protein aggregates. Chaperones favour the native folding of proteins either as "holdases", sequestering hydrophobic regions in misfolding polypeptides, and/or as "unfoldases", forcibly unfolding and disentangling misfolded polypeptides from aggregates. Whereas in bacteria, plants and fungi Hsp70/40 acts in concert with the Hsp100 (ClpB) unfoldase, Hsp70/40 is the only known chaperone in the cytoplasm of mammalian cells that can forcibly unfold and neutralize cytotoxic protein conformers. Owing to its particular spatial configuration, the bulky 70 kDa Hsp70 molecule, when distally bound through a very tight molecular clamp onto a 50-fold smaller hydrophobic peptide loop extruding from an aggregate, can locally exert on the misfolded segment an unfolding force of entropic origin, thus destroying the misfolded structures that stabilize aggregates. ADP/ATP exchange triggers Hsp70 dissociation from the ensuing enlarged unfolded peptide loop, which is then allowed to spontaneously refold into a closer-to-native conformation devoid of affinity for the chaperone. Driven by ATP, the cooperative action of Hsp70 and its co-chaperone Hsp40 may thus gradually convert toxic misfolded protein substrates with high affinity for the chaperone, into non-toxic, natively refolded, low-affinity products. Stress- and mutation-induced protein damages in the cell, causing degenerative diseases and aging, may thus be effectively counteracted by a powerful network of molecular chaperones and of chaperone-related proteases.

The structure of a globular protein is known to be affected by addition of acids or alkalis. The fact that a protein unfolds or at least denatures in solutions with extreme pH values was known for a long time. Prof. Anthony L. Fink (Tony) brought this field to a new level by showing that acid-unfolded proteins can partially regain their ordered structures in the presence of various anions. This review analyses his contributions to this branch of protein science. His studies provided an explanation for the molecular mechanisms underlying anion-induced refolding of acid-unfolded proteins. He was the first who clearly showed that different anions differed dramatically in their efficiency to bring about this refolding. This difference was shown to be manifested in the amounts of anions needed to complete a structural transition, in the degree of cooperativity of these transitions, and in the amounts of ordered structure induced in the acid-unfolded proteins at the completion of the corresponding transitions. He was also first who undoubtedly demonstrated that, at low pH, proteins can populate discrete partially folded conformations in the presence of different anions. His papers are highly cited, clearly showing that the work of Tony Fink on anion-induced folding of globular proteins made a great impact to the protein folding field.

Current research suggests that the function of the prion protein (PrP) is linked to its ability to bind copper. PrP is implicated in copper regulation, copper buffering and copper-dependent signaling. Moreover, in the development of prion disease, copper may modulate the rate of protein misfolding. PrP possesses a number of copper sites, each with distinct chemical characteristics. Most studies thus far have concentrated on elucidating chemical features of the octarepeat region (residues 60-91, hamster sequence), which can take up to four equivalents of copper, depending on the ratio of Cu2+ to protein. However, other sites have been proposed, including those at histidines 96 and 111, which are adjacent to the octarepeats, and also at histidines within PrP's folded C-terminal domain. Here, we review the literature of these copper sites extrinsic to the octarepeat region and add new findings and insights from recent experiments.

Protein aggregation, being one of the hottest topics of modern protein science, is recognized now as a serious biomedical and biotechnological problem. Protein aggregation is considered as a causative factor (or at least an associated symptom) of a wide spectrum of human pathologies. Furthermore, aggregation and precipitation are known to trammel recombinant protein production, as well as to affect the manufacture, storage and delivery of proteinaceous drugs. Therefore, this topic attracts the serious attention of many researchers, a conclusion that follows from the average daily publication of 7-8 scientific papers dedicated to the various aspects of protein aggregation. However, the situation was rather different 15-20 years ago when it was believed that the formation of protein aggregates causing the irreversibility of unfolding or denaturation of some proteins was nothing more than an annoying experimental artifact, hampering the detailed characterization of the unfolding/denaturation processes. At that time, only a few laboratories (including the laboratory of Prof. Anthony L. Fink) seriously worked on understanding the molecular mechanisms of this "artifact". In this review, we summarize some of the early work of Tony Fink on aggregation, of protein folding intermediates and on the analysis of the structural consequences of this process.

Eg5 is a kinesin whose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetry of our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5'-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP-Eg5 complexes using a thermal shift assay. We measured up to a 7 degrees C increase in the thermal melting (T(m)) of Eg5 for an inhibitor that produced IC(50) values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase (ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assays correlated with the magnitude of the thermal stability enhancement of ADP-Eg5. The thermal shift assay also confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assay is applicable to a range of chemotypes and can be useful in evaluating both potent (nM) and relatively weakly binding (microM) leads. Overall, the thermal shift assay was found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assay screens by providing an alternative determination of inhibitor potency.

The significant increase in the demand for purified protein for crystallization and structural studies has made necessary the development of multi-sample methods for identifying solution conditions that affect protein stability and aggregation. Conditions that stabilize proteins can improve protein purification and crystallization. These methods can be used to identify small molecule compounds or inhibitors that interact with the purified proteins, and might serve as starting points for drug discovery. In this article three methods for measuring protein stability and aggregation are described and discussed: differential scanning fluorimetry (DSF), differential static light scattering (DSLS), and isothermal denaturation (ITD).

This research work allows a direct comparison between collagen solutions of equal concentration derived from the two widely used collagen sources: bovine Achilles tendon (BAT) and rat tail tendon (RTT), and extraction methods: acid (AS) and pepsin (PS) solubilization on the properties of extruded collagen fibers. Scanning electron microscopy revealed that the substructure of the collagen fibers was the same independent of the treatment. Transmission electron microscopy revealed that the AS collagen-derived fibers were comprised of thick quarter-staggered fibrils, while the coexistence of thin nonbanded and thick banded fibrils was apparent for the PS collagen-derived fibers. The BAT-derived fibers demonstrated higher denaturation temperature than the RTT-derived ones (p < 0.05). The extraction method had no influence on the thermal characteristics of the fibers produced (p > 0.05). ASBAT collagen was of higher viscosity than both ASRTT and PSBAT (p < 0.002), and therefore larger diameter fibers were obtained (p < 0.001). An inversely proportional relationship between dry-fiber diameter and stress at break was observed within the treatments. The PS yielded 10 times more soluble collagen from BAT and the derived fibers were of similar tensile strength, stiffness, and elongation (p > 0.05) as those derived from the AS collagen. No significant difference was observed for the stress at break for the ASBAT and the ASRTT, while significant difference was observed for the elongation and modulus values (p < 0.005). Overall, reconstituted collagen fibers were produced with properties similar to native or synthetic fibers to suit a wide range of tissue engineering applications.

Resolving the complete folding pathway of a protein is a major challenge to conventional experimental methods because of the rapidity and complexity of folding. Here, we show that entrapment of the protein cytochrome c in wet, optically transparent, porous silica gel matrices has enabled a dramatic expansion, to days or weeks, of the folding time, allowing direct observation of the entire folding pathway using a combination of three spectroscopic techniques, far-ultraviolet circular dichroism, tryptophan fluorescence, and Soret absorption spectroscopy. During refolding in silica gels, collapse and helix formation occur in a stepwise manner, as observed in aqueous solution. Analysis of kinetics and transient spectra indeed reveals a sequence of four distinct intermediates with progressively increasing degrees of folding, two of which closely resemble those previously characterized in solution, namely, the early collapsed and the molten globule intermediates. The other two are the precollapsed and pre-molten globule intermediates that may escape detection by conventional kinetic methods. Interestingly, varying the strength of the gel network has a dramatic effect on the folding time, but no significant effect on the structural features of each folding intermediate, indicating that the interaction between the protein and gel matrix has no measurable effect on the folding pathway. These results better define the major pathway of cytochrome c folding. In addition, in this paper we present the results of the application of this method to a simple, apparent two-state folder ubiquitin, helping to interpret the results for cytochrome c.

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells (and by extension, that of the multicellular organisms they form) is the result of cycles of mechanosensing, mechanotransduction, and mechanoresponse. Recently developed single-molecule atomic force microscopy techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins. Single-molecule mechanical techniques are providing fundamental information on the structure and function of proteins and are becoming an indispensable tool to understand how these molecules fold and work.

Evolution has tuned membrane proteins to exist in a lipid bilayer, provide for cell-cell communication, transport solutes, and convert energies. These proteins exhibit a hydrophobic belt that interacts with the lipid bilayer. Detergents are therefore used to extract membrane proteins and keep them in solution for purification and subsequent analyses. However, most membrane proteins are unstable when solubilized and hence often not accessible to NMR or X-ray crystallography. The atomic force microscope (AFM) is a powerful tool for imaging and manipulating membrane proteins in their native state. Superb images of native membranes have been recorded, and a quantitative interpretation of the data acquired using the AFM tip has become possible. In addition, multifunctional probes to simultaneously acquire information on the topography and electrical properties of membrane proteins have been produced. This progress is discussed here and fosters expectations for future developments and applications of AFM and single-molecule force spectroscopy.

Although protein-folding studies began several decades ago, it is only recently that the tools to analyze protein folding at the single-molecule level have been developed. Advances in single-molecule fluorescence and force spectroscopy techniques allow investigation of the folding and dynamics of single protein molecules, both at equilibrium and as they fold and unfold. The experiments are far from simple, however, both in execution and in interpretation of the results. In this review, we discuss some of the highlights of the work so far and concentrate on cases where comparisons with the classical experiments can be made. We conclude that, although there have been relatively few startling insights from single-molecule studies, the rapid progress that has been made suggests that these experiments have significant potential to advance our understanding of protein folding. In particular, new techniques offer the possibility to explore regions of the energy landscape that are inaccessible to classical ensemble measurements and, perhaps, to observe rare events undetectable by other means.

Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.

We report the areal growth kinetics of fibrinogen adsorbed on model hydrophobic and hydrophilic surfaces measured via an adsorption probe method. This approach exploits the adsorption of probe molecules to determine the evolution of fibrinogen test molecules under conditions where the fibrinogen test molecules adsorb at relatively dilute surface conditions, minimizing interactions between them. It is found that fibrinogen test molecules spread from an average initial footprint of 100 nm2 to a final footprint near 500 nm2 per molecule on the hydrophobic surface, with a single-exponential decay of 1735 s. On a hydrophilic monolayer, the area increases from 100 to 160 nm2 with a characteristic time of 6740 s. These results demonstrate the power of the adsorption probe approach and comprise the first measurements of the averaged area relaxations of adsorbed proteins. The observation of single-exponential dynamics is remarkable, given the extensive relaxation on the hydrophobic surface, which must involve fibrinogen denaturing.

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.

Dimethylglycine dehydrogenase (Me(2)GlyDH) is a mitochondrial enzyme that catalyzes the oxidative demethylation of dimethylglycine to sarcosine. The enzyme requires flavin adenine dinucleotide (FAD), which is covalently bound to the apoprotein via a histidyl(N3)-(8alpha)FAD linkage. In the present study, the mature form of rat Me(2)GlyDH has been over-expressed in Escherichia coli as an N-terminally 6-His-tagged fusion protein. The over-expressed protein distributed almost equally between the soluble and insoluble (inclusion bodies) cell fraction. By applying the soluble cell lysate to a nickel-chelating column, two fractions were eluted, both containing a nearly homogeneous protein with a molecular mass of 93 kDa, on SDS-PAGE. The first protein fraction was identified by Western blotting analysis as the covalently flavinylated Me(2)GlyDH. It showed optical properties and specific activity (240 nmol/min/mg protein) similar to those of the native holoenzyme. The second fraction was identified as an underflavinylated (apo-) form of Me(2)GlyDH, with a 70% lower specific activity. The recombinant holoenzyme exhibited optimal activity at pH 8.5, an activation energy of about 80 kJ/mol, and two KM values for N,N-dimethylglycine (KM1 = 0.05 mM and KM2 = 9.4 mM), as described for the native holoenzyme. Starting from the inclusion bodies, the unfolded flavinylated enzyme was solubilized by SDS treatment and refolded by an 80-fold dilution step, with a reactivation yield of 50-60%.

We used a temperature-jump isothermal denaturation procedure with various methods of detection to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection-ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism-as well as differential scanning calorimetry were used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence were used to quantitate the dose dependencies of the ligand effects. We found that the first step in the denaturation of MurB is the rapid loss of flavin from the active site and that the two chemical inhibitors appeared to destabilize the interaction of the cofactor with the enzyme but stabilize the global unfolding. The kinetics of the denaturation process as well as the loss of flavin fluorescence on binding established that both compounds had nanomolar affinities for the enzyme. We showed that coupling of the various detection methods with isothermal denaturation yields a powerful regimen to provide analytical data for assessing inhibitor specificity for a protein target.

A new method to examine the structure and stability of proteins using footprinting is applied to examine the unfolding of apomyoglobin. Unlike previous cleavage based footprinting methods, synchrotron X-ray protein footprinting is based on a quantitative determination of the extent and the site of radiolytic modification of amino acid side chains, analyzed using mass spectrometry. The amino acids most susceptible to radiolytic oxidation (cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline, and leucine) serve as convenient probes of protein structure to monitor changes in solvent accessibility. To determine if the technique can measure quantitative properties of proteins relevant to structure and function, we examined the equilibrium unfolding of apomyoglobin in urea and compared the results to data derived from fluorescence studies under the same conditions., (Copyright 2001 Academic Press.)

Ribosomal p90rsk is a kinase of central importance in transducing mitogenic signals from an activated receptor to the cell nucleus and for protein synthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunological analyses (immunoblotting and immunoprecipitation); (ii) enzyme activity assays (in vitro and "in-gel"); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/"in-gel" assay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90rsk kinase activity "in-gel", based on a renatured-protein method that allows for the direct quantitation of enzyme activity. Finally, we present an algorithm that can be readily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli. In our case, it was found that activation of p90rsk was higher in proliferating leukemic cells than in mature neutrophils, indicating that a suppression of key signal transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90rsk could be easily extrapolated to the study of any signal transduction molecule, provided it has a phosphotransferase activity.

A method to release L-asparaginase (EC 3.5.1.1) from ATCC Escherichia coli 11303 cells by chemical permeabilization was studied. It was found that a combination of K2HPO4 and Triton X100 was effective. The influences of K2HPO4 concentration, Triton concentration, E. coli cell concentration and pH on the release of enzyme and proteins were investigated in detail. Experimental results showed that 12.5% (w/v) K2HPO4, 2% (w/v) Triton X100 and 3 x 10(8) cells/mL made the amount of enzyme released over 70%. L-Asparaginase in K2HPO4 and Triton solution could remain stable at least for 24 h. The release effect of K2HPO4 and Triton X100 used simultaneously was better than that of K2HPO4 and Triton X100 used separately in succession. Electron microscopy indicated that the chemical treatment altered the surface structure of E. coli cells but did not break them. As the method does not produce a large amount of cell fragments and the amount of enzyme released is relatively high, it can be thought to be an valuable and economic method to release intracellular enzyme.

The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.

To test the validity of thermodynamic parameters from the equilibrium method, we have studied the reversible heat-induced denaturations of lysozyme, ribonuclease A, cytochrome c and myoglobin at various pH values, using absorption spectral measurements. For each protein, if a linear temperature-dependence of the pre- and post-transition baselines is assumed for the analysis of the conformational-transition curve, the estimate of DeltaH (the enthalpy change on denaturation at T(m), the midpoint of denaturation) is significantly less than DeltaH, the value obtained by the calorimetric measurements. If the analysis of thermal-denaturation curves assumes that the temperature-dependence of pre- and post-transition baselines is described by a parabolic function, there exists an excellent agreement between DeltaH(m) values of all proteins obtained from equilibrium and calorimetric methods. The latter analysis is supported by the studies on model compounds, for measurements of absorption properties of tyrosine, tryptophan and haem as a function of temperature suggested that the temperature-dependencies of the optical properties are indeed non-linear. We have observed that for each protein the constant-pressure heat-capacity change on denaturation (DeltaC(p)) determined from the plots of DeltaH versus T(m) is not only independent of the method of analysis of the transition curve, but it is also in excellent agreement with calorimetric DeltaC(p). An important conclusion of this study is that for these proteins that exhibit two-state character, all stability parameters are measured with the same error as that observed with a calorimeter.

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates., (Copyright 1998 John Wiley & Sons, Inc.)

A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a 19F probe attached to the beta-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mM in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.

In the 4 years since Hammes (87) reviewed, in this journal, the application of flow and relaxation techniques to the study of primary processes in chemical reactions these methods have been applied to a wide range of biochemical problems. These applications have been facilitated by advances in stopped flow and temperature jump instrumentation, especially with respect to the range of physical properties that may be observed during a reaction, and the availability of instruments.

1. The three main components of the 1.5-2s ultracentrifugal peak of carp myogen (white muscle) have been isolated by ammonium sulphate fractionation and zone electrophoresis, and crystallized. 2. The molecular weights of these three proteins were determined by sedimentation and diffusion, by the Archibald method and by amino acid analysis, and found to lie between 9000 and 13000. 3. Their complete amino acid compositions were determined by column chromatography and by their ultraviolet spectra. Both methods revealed abnormal compositions, including the absence of tryptophan and methionine and the presence of large amounts of phenylalanine. At most 1 residue each of tyrosine, cysteine, proline, arginine and histidine was found/molecule. 4. The specific viscosity of component 3 was lower than that of other small globular proteins described so far, a fact that suggests that these proteins approximate more closely to the ideal case of the spherical protein molecule. Also, the presence of a single residue of several amino acids, the absence of disulphide bonds, and the apparent reversibility of denaturation by urea of component 3 suggest that the study of these molecules could provide new information on the structure of proteins.