Protein unfolding during reversed-phase chromatography: I. Effect of surface properties and duration of adsorption.

Residue-level features of bovine pancreatic trypsin inhibitor (BPTI) unfolding on reversed-phase chromatography (RPC) surfaces were investigated using hydrogen-deuterium exchange labeling and NMR. A set of silica-based RPC surfaces was used to examine the influence of alkyl chain length and media pore size on adsorbed BPTI conformation. In all cases there was substantial unfolding in the adsorbed state; however, residual protection from exchange was consistently observed. Particle pore size did not influence conformation substantially for C4-alkyl modified silica; however, 120 A pore C18 media produced more hydrogen exchange than any other surface examined. In this case, the radius of curvature inside the pore approaches the size of the BPTI molecule. Generally, the pattern of hydrogen exchange protection was uniform; however, the beta-sheet region was selectively protected on the large-pore C18 media. The beta-sheet region forms a hydrophobic core that forms early when BPTI folds in solution. This suggests that partially unfolded states possessing a native-like structure play an important role in adsorption and elution in RPC. Finally, increased contact time with the surface before elution fostered unfolding and altered chromatographic behavior considerably.     

Mobile phase modifier effects in multimodal cation exchange chromatography

This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.     

Protein instability during HIC: describing the effects of mobile phase conditions on instability and chromatographic retention

Hydrophobic interaction chromatography (HIC) is known to be potentially denaturing to proteins, but the effects of mobile phase conditions on chromatographic behavior are not well understood. In this study, we apply a model describing the effects of secondary protein unfolding equilibrium on chromatographic behavior, including the effects of salt concentration on both stability and adsorption. We use alpha-lactalbumin as a model protein that in the presence and absence of calcium, allows evaluation of adsorption parameters for folded and unfolded species independently. The HIC adsorption equilibrium under linear binding conditions and solution phase protein stability have been obtained from a combination of literature and new experiments. The effect of salt concentration on protein stability and the rate constant for unfolding on the chromatographic surface have been determined by fitting the model to isocratic chromatography data under marginally stable conditions. The model successfully describes the effects of added calcium and ammonium sulfate. The results demonstrate the importance of considering the effects on stability of mobile phase modifiers when applying HIC to marginally stable     

Adhesion of Azospirillum brasilense to polystyrene: Influence of ionic strength through protein adsorption

The influence of ionic strength on the adhesion of Azospirillum brasilense to polystyrene has been examined by comparing water and phosphate buffer saline (PBS) as suspending media. Polystyrene supports analysed by X‐ray photoelectron spectroscopy (XPS) after adhesion in PBS for 2 h or 24 h and detachment of adhering cells showed a higher protein surface concentration, reflected by the N/C atomic concentration ratio, compared to supports analysed after adhesion in water. It was shown that PBS both favours protein release by the cells into the solution and enhances the tendency of proteins to adsorb at the support surface.

After 2 h contact time, the increase in the concentration of adsorbed proteins in PBS was related to an increase in adhesion density. However, the observation that the adhesion density after 24 h was lower in PBS than in water indicated that the amount of proteins adsorbed at the support surface controls cell adhesion in a complex way. In PBS, a thick layer of proteinaceous material retaining the bacterial cells is formed; this leads to underestimation of the density of adhering cells as well as to a heterogeneous adhesion pattern and to a relatively low adhesion density due to detachment of pellicles upon rinsing.

The ionic strength thus influences bacterial adhesion in a more subtle way than simply through double layer interactions between the cells and the support.     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Protein instability during HIC: hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects

Unfolding of marginally stable proteins is a significant factor in commercial application of hydrophobic interaction chromatography (HIC). In this work, hydrogen-deuterium isotope exchange labeling has been used to monitor protein unfolding on HIC media for different stationary phase hydrophobicities and as a function of ammonium sulfate concentration. Circular dichroism and Raman spectroscopy were also used to characterize the structural perturbations experienced by solution phase protein that had been exposed to media and by protein adsorbed on media. As expected, greater instability is seen on chromatographic media with greater apparent hydrophobicity. However, increased salt concentrations also led to more unfolding, despite the well-known stabilizing effect of ammonium sulfate in solution. A thermodynamic framework is proposed to account for the effects of salt on both adsorption and stability during hydrophobic chromatography. Using appropriate estimates of input quantities, analysis with the framework can explain how salt effects on stability in chromatographic systems may contrast with solution stability.     

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.     

Protein instability during HIC: Evidence of unfolding reversibility,and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

A two‐conformation, four‐state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four‐state model to the effect of salt concentration on the adsorption and unfolding of the model protein α‐lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild‐type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of α‐lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild‐type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (～6–8 kJ/mol) due to increased instability of the protein. Finally, the four‐state model is used to accurately predict the apparent adsorption strength of a disulfide bond‐reduced variant. Biotechnol. Bioeng. 2009;102: 1416–1427. © 2008 Wiley Periodicals, Inc.     

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR*

The susceptibility of the cellular prion protein (PrP^C) to convert to an alternative misfolded conformation (PrP^Sc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.     

The solution structure and dynamics of the DH‐PH module of PDZRhoGEF in isolation and in complex with nucleotide‐free RhoA

The DH‐PH domain tandems of Dbl‐homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho‐family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH‐PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small‐angle X‐ray scattering (SAXS), and hydrogen‐deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH‐PH tandem of RhoA‐specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH‐PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide‐free RhoA exhibits elevated dynamics when in complex with DH‐PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.     

The interaction of proteins with hydroxyapatite: II. Role of acidic and basic groups

The elution behavior from hydroxyapatite columns of the modification products of seven basic and three acidic proteins has been investigated. Three classes of NH₂ derivatives were prepared. These consisted of (1) replacement by a guanidyl group with no change in charge; (2) blocking with loss of charge; and (3) replacement of positive charges by negative ones. Two types of COOH derivatives were prepared: (1) blocking with loss of charge; and (2) replacement of COOH by SO₃H with no change in charge. The elution behavior of the derivatives in PO₄, F^?, Cl^?, ClO₄^?, and Ca²⁺ ion eluants showed that (1) the elution patterns are determined by the isoelectric points of the proteins, there being no symmetry between the binding or elution behavior of acidic and basic proteins; (2) the binding of basic proteins requires the presence of a high density of positively charged groups; (3) the binding of all proteins to hydroxyapatite equilibrated with phosphate buffer is enhanced by a decrease in the number of their negative charges; and (4) calcium ions affect the binding of proteins to hydroxyapatite at the level of carboxyls, since clusters of carboxyls strengthen both the interaction with Ca²⁺ and the binding to hydroxyapatite.     

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

Conformational Ensemble and Biological Role of the TCTP Intrinsically Disordered Region: Influence of Calcium and Phosphorylation

The translationally controlled tumor protein (TCTP) is a multifunctional protein that may interact with many other biomolecules, including itself. The experimental determinations of TCTP structure revealed a folded core domain and an intrinsically disordered region, which includes the first highly conserved TCTP signature, but whose role in the protein functions remains to be elucidated. In this work, we combined NMR experiments and MD simulations to characterize the conformational ensemble of the TCTP intrinsically disordered loop, in the presence or not of calcium ions and with or without the phosphorylation of Ser46 and Ser64. Our results show that these changes in the TCTP electrostatic conditions induce significant shifts of its conformational ensemble toward structures more or less extended in which the disordered loop is pulled away or folded against the core domain. Particularly, these conditions impact the transient contacts between the two highly conserved signatures of the protein. Moreover, both experimental and theoretical data show that the interface of the non-covalent TCTP dimerization involves its second signature which suggests that this region might be involved in protein–protein interaction. We also show that calcium hampers the formation of TCTP dimers, likely by favoring the competitive binding of the disordered loop to the dimerization interface. All together, we propose that the TCTP intrinsically disordered region is involved in remodeling the core domain surface to modulate its accessibility to its partners in response to a variety of cellular conditions.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

P-31 Nuclear Magnetic Resonance Analysis of Brain: II. Effects of Oxygen Deprivation on Isolated Perfused and Nonperfused Rat Brain

Phosphatic metabolite (perchloric acid extractable) concentrations of cerebral tissues were analyzed by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy following external perfusion of the isolated rat brain (30 min or 60 min) under the following conditions: (a) constant perfusion pressure with either fluorocarbon- or erythrocyte-based medium, and (b) constant perfusate flow rate (3 ml/min) with the erythrocyte-based medium. Metabolite concentrations of control perfused brains were compared with those in nonperfused controls to provide a basis for detecting any qualitative or quantitative changes in cerebral metabolite composition. Metabolic responses of perfused brains to ischemia (incomplete ischemia, 83% reduction in flow for 10 min; transient complete ischemia for 1.5 or 2 min) were evaluated immediately after the ischemic episode and at selected time points during reperfusion (3 and 15 min). Alterations in cerebral metabolite levels induced by hypoxia were analyzed using a nonperfused rat brain model. Irrespective of the perfusion method employed, the phosphatic metabolites of control perfused rat brains were identical quantitatively to those of the nonperfused controls. Cerebral ischemia resulted in significantly increased levels of ADP, AMP + IMP, Pi, fructose 1,6-diphosphate, and glycerol 3-phosphate (global ischemia only), whereas ATP and phosphocreatine (PCr) levels declined significantly. The magnitude of these changes varied with the severity of the ischemia; however, following 15 min of control reperfusion metabolite levels had reverted to preischemic values. Significant perturbations in tissue phosphoethanolamine (3.84 delta resonance) content were evident at various time points during ischemia and postischemic recovery, which varied according to the perfusion conditions. In contrast to the changes observed in response to ischemia, hypoxia affected only cerebral high-energy phosphate levels. ATP and PCr levels were reduced, while a concomitant, essentially equimolar, increase in Pi and ADP was observed. The present studies indicate that in terms of phosphatic metabolites, the control equilibrated isolated perfused rat brain is quantitatively and qualitatively indistinguishable from the nonperfused rat brain in vivo regardless of the perfusion conditions (constant flow versus constant pressure). The metabolic responses to ischemia and hypoxia, as measured by P-31 NMR, were consistent with the pattern of changes reported elsewhere. Overall, P-31 NMR spectroscopic evaluation of the intact rat brain provides a potential experimental context for dynamic measures of cerebral metabolism under exogenously controlled conditions. Th     

1H-N.m.r. and c.d. investigation of the histidine side chain conformation in small peptides

Three series of model peptides containing histidine have been examined by ¹H-n.m.r. and c.d. spectroscopy: X-His peptides with X = Gly, Ala, Leu; His-X peptides with X = Gly, Ala, Leu, Ser, Lys, Phe, Tyr; and Pro-His-X peptides with X = Gly; Ala; Leu; Val; Phe; Tyr, C.d. spectra were obtained for pH values between 1 and 11 to give titration curves [θ] vs. pH; ¹H-n.m.r. spectra were recorded at four selected pH values corresponding to defined ionic species. ¹H-n.m.r. spectra in Me₂SO of the NH₃⁺, Imid⁺, COO^? ionic state (pH 4.5) were also obtained. The histidine side chain conformation in the various peptides and the changing ionic states is reflected in the ³J_αβ,β′ coupling constants, the Δδ ββ′ anisochrony values and the c.d. histidine chromophore contribution at 215 nm, and qualitative and semiquantitative correlations can be established between these parameters. Whereas the histidine side chain conformation is quite different in each of the three series, and varies with the ionic state and environment, it is practically identical for each peptide within a series: the nature of the X-residue does not exert any influence on the histidine side chain conformational behaviour. Thus, the classical rotamer distribution R I > R II > R III which is due to steric factors is usually observed unless specific intramolecular interactions such as hydrogen or ionic bonds override these.     

Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides.

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC¹]-Gm; [TOAC¹, Ser^2,6,11,15]-Gm; [Trp⁷]-Gm; [Ser^2,6,11,15, Trp⁷]-Gm; [Trp⁹]-Gm; and [Ser^2,6,11,15, Trp⁹]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a β-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC¹]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.