Characterization of the solution conformations of leuprolide acetate.

Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt), a potent LHRH agonist in wide clinical use, was characterized conformationally by NMR and circular dichroism. It displayed quite different preferred conformations under different solution conditions: two low population beta-turns in water, a nascent helix in TFE/water at low pH, and a high population beta-turn in TFE/water at slightly acidic pH. The pH-related conformational change in TFE/water is attributed to the pK(a) of the acetate counterion, not to ionizable groups on the peptide. None of these conformations are in exact agreement with previous computational predictions.

The effects of Tween 20 and sucrose on the stability of anti-L-selectin during lyophilization and reconstitution.

We have chosen an anti-L-selectin antibody as a model protein to investigate the effects of sucrose and/or Tween 20 on protein stability during lyophilization and reconstitution. Native anti-L-selectin secondary structure is substantially retained during lyophilization in the presence of sucrose (1 or 0.125%). However, aggregation of the protein during reconstitution of lyophilized protein powders prepared without sucrose is not reduced by the presence of sucrose in the reconstitution medium. Aggregate formation upon reconstitution is completely inhibited by freeze drying the protein with sucrose and reconstituting with a 0.1% Tween 20 solution. Tween 20 (0.1%) also partially inhibits loss of native anti-L-selectin secondary structure during lyophilization. However, upon reconstitution the formulations lyophilized with Tween 20 contain the highest levels of aggregates. The presence of Tween in only the reconstitution solution appears to inhibit the transition from dimers to higher order oligomers. Potential mechanism(s) for the Tween 20 effects were investigated. However, no evidence of thermodynamic stabilization of anti-L-selectin conformation (e.g., by Tween 20 binding) could be detected.

Controlling deamidation rates in a model peptide: effects of temperature, peptide concentration, and additives.

The rate of deamidation of the Asn residue in Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA), a model peptide, was determined at pH 9 (400 mM Tris buffer) as a function of temperature and peptide concentration. Over the temperature range 5-65 degrees C, deamidation followed Arrhenius behavior, with an apparent activation energy of 13.3 kcal/mol. Furthermore, increasing the peptide concentration slows the rate of deamidation. Self-stabilization with respect to deamidation has not been reported previously. The rate of deamidation was also determined in the presence of sucrose and poloxamer 407 (Pluronic F127). In both cases, the rate of deamidation was retarded by up to 40% at 35 degrees C. In aqueous solutions containing poloxamer 407, the degree of stabilization is independent of formation of a reversible thermosetting gel. With sucrose, maximum reduction in the deamidation rate was attained with as little as 5% (w/v). Addition of sucrose results in a greater conformational preference for a type II beta-turn structure, which presumably is less prone to intramolecular cyclization and subsequent deamidation.

Counteracting effects of renal solutes on amyloid fibril formation by immunoglobulin light chains.

In primary (light chain-associated) amyloidosis, immunoglobulin light chains deposit as amyloid fibrils in vital organs, especially the kidney. Because the kidney contains high concentrations of urea that can destabilize light chains as well as solutes such as betaine and sorbitol that serve as protein stabilizers, we investigated the effects of these solutes on in vitro amyloid fibril formation and thermodynamic stability of light chains. Two recombinant light chain proteins, one amyloidogenic and the other nonamyloidogenic, were used as models. For both light chains, urea enhanced fibril formation by reducing the nucleation lag time and diminished protein thermodynamic stability. Conversely, betaine or sorbitol increased thermodynamic stability of the proteins and partially inhibited fibril formation. These solutes also counteracted urea-induced reduction in protein thermodynamic stability and accelerated fibril formation. Betaine was more effective than sorbitol. A model is presented to explain how the thermodynamic effects of the solutes on protein state equilibria can alter nucleation lag time and, hence, fibril formation kinetics. Our results provide evidence that renal solutes control thermodynamic and kinetic stability of light chains and thus may modulate amyloid fibril formation in the kidney.

Comparative fourier transform infrared and circular dichroism spectroscopic analysis of alpha1-proteinase inhibitor and ovalbumin in aqueous solution.

Alpha1-proteinase inhibitor (alpha1Pi) and ovalbumin are both members of the serpin superfamily. They share about a 30% sequence identity and exhibit great similarity in their three-dimensional structures. However, no apparent functional relationship has been found between the two proteins. Unlike alpha1Pi, ovalbumin shows no inhibitory effect to serine proteases. To see whether or not a conformational factor(s) may contribute to the functional difference, we carried out comparative analysis of the two proteins' secondary structure, thermal stability, and H-D exchange using FT-IR and CD spectroscopy. FT-IR analysis reveals significant differences in the amide I spectral patterns of the two proteins. Upon thermal denaturation, both proteins exhibit a strong low-wavenumber beta-sheet band at 1624 cm(-1) and a weak high-wavenumber beta-sheet band at 1694 cm(-1), indicative of intermolecular aggregate formation. However, the midpoint of the thermal-induced transition of alpha1Pi (approximately 55 degrees C) is 18 degrees C lower than that of ovalbumin (approximately 73 degrees C). The thermal stability analysis provides new insight into the structural changes associated with denaturation. The result of H-D exchange explains some puzzling spectral differences between the two proteins in D2O reported previously.

Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor.

Metal-induced precipitation of protein therapeutics is being used and further developed as a processing step in protein formulation and may have utility in protein purification and bulk storage. In such processes, it is imperative that native protein structure is maintained and the metal complexation is reversible. In the current study, we investigated the effects of zinc-induced precipitation on recombinant human growth hormone (rhGH) and recombinant human nerve growth factor (rhNGF). On the addition of ethylenediaminetetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific metal binding sites and require specific molar ratios of zinc to protein to initiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc complexes and zinc-induced precipitates, as measured by infrared and circular dichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiary structural alterations, whereas zinc binding did not alter the tertiary structure of rhNGF. These studies indicated that metal-induced precipitation provides a method to maintain proteins in their native state in precipitates, which may be useful for purification, storage, and formulation.

Human immunodeficiency virus-related risk behavior among African-American females.

This study draws attention to the demographic shift in the population of HIV-infected African Americans from young, low-income, unmarried homosexual, and injecting drug users to female, heterosexual, higher income, and older persons. We used data from the 1995 Survey of Family Growth, sponsored by the National Center for Health Statistics, to examine the patterns of HIV-related risk behavior (consistent condom use, number of sexual partners, sex education in birth control methods) among African-American females. We found that only 33.3% of the African-American females had indicated that their partners always used condoms; 23.8% had seven or more lifetime sexual partners; and nearly 30% did not have any sex education in birth control methods, sexually transmitted diseases, or abstinence. In addition, African-American females who had partners who had not used condoms in the last 12 months were less likely than those who reported occasional condom use to perceive that they were infected with HIV (21.1% vs. 33.1%). These risk factors were prevalent among low-income African-American females with low socioeconomic status (SES) as well as black women with higher SES who lived in smaller cities and suburbs. These results highlight the need for HIV prevention strategies that cut across socioeconomic class, gender, sexual orientation, and place of residence.

Stability of human serum albumin during bioprocessing: denaturation and aggregation during processing of albumin paste.

PURPOSE: To assess the impact of various bioprocessing steps on the stability of freshly precipitated human serum albumin (HSA) obtained from pooled human plasma. METHODS: After initial precipitation of HSA from plasma, the resultant paste is either (a) lyophilized or (b) washed with acetone and then air-dried in order to obtain a dry powder. The structure of HSA was examined using Fourier transform infrared (IR) spectroscopy. The extent of aggregation of redissolved HSA was measured using both dynamic light scattering and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Both lyophilization and air-drying perturb the secondary structural composition of HSA, as detected by infrared (IR) spectroscopy. Upon dissolution of dried paste, most of the protein refolds to a native-like conformation. However, a small fraction of the protein molecules form soluble aggregates that can be detected by both dynamic light scattering and SDS-PAGE. The level of aggregation is so low that it could not be detected in the bulk by either circular dichroism or IR spectroscopy. The lyophilized protein, which appears to be more unfolded in the solid state than the acetone washed/air-dried material, exhibits a higher level of aggregation upon dissolution. CONCLUSIONS: There is a direct correlation between the extent of unfolding in the solid state and the amount of soluble aggregate present after dissolution. Moreover, the presence of the aggregates persists throughout the remainder of the purification process, which includes dissolution, chromatography, sterile filtration and viral inactivation steps. Analytical methods used to monitor the stability of biopharmaceuticals in the final product can be used to assess damage inflicted during processing of protein pharmaceuticals.

Long chain arginine esters: a new class of cationic detergents for preparation of hydrophobic ion-paired complexes.

The ability of stoichiometric amounts (based on charged groups) of ionic detergents to bind to oppositely charged ionic compounds has been recently reviewed. These hydrophobic ion-paired (HIP) complexes display altered solubility properties. Most of the work to date on HIP compelxes has focused on basic drugs and anionic detergents. It would be extremely useful to extend this approach to acidic compounds, including DNA and RNA. However, most cationic detergents are relatively toxic. It is hypothesized that detergents constructed from naturally occurring or well tolerated components, coupled by labile linkages, will be less toxic and still able to form strong HIP complexes. This study describes the synthesis and characterization of long chain alkyl esters of arginine. This class of cationic detergents, which have not been reported previously, are less cytotoxic than alkyltrimethylammonium detergents, possibly making them more acceptable in drug delivery applications. These arginine esters exhibit detergent-like properties. For example, the dodecyl ester of arginine has a critical micelle concentration of 0.07 mM, while being approximately 5-10 fold less toxic than tetradecyltrimethylammonium bromide. The arginine dodecyl ester forms stable HIP complexes with plasmid DNA. The complex is sufficiently stable to allow some modest level of transfection with Cos-7 cells in a time- and concentration-dependent fashion. This work demonstrates that arginine-based cationic detergents are effective ion-pairing agents, appear to be less toxic than alkyltrimethylammonium compounds, and form stable complexes with DNA.

Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran.

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting T(g) of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and T(g) by freeze-drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T(g) observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability.

Thermodynamic modulation of light chain amyloid fibril formation.

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.

Domain structure of gpNu1, a phage lambda DNA packaging protein.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of a dsDNA genome into the confines of the viral capsid. The holoenzyme is composed of gpA and gpNu1 subunits in a gpA(1) x gpNu1(2) stoichiometry. While genetic studies have described regions within the two proteins responsible for DNA binding, capsid binding, and subunit interactions in the holoenzyme complex, biochemical characterization of these domains is limited. We have previously described the cloning, expression, and biochemical characterization of a soluble DNA binding domain of the terminase gpNu1 subunit (Met1 to Lys100) and suggested that the hydrophobic region spanning Lys100 to Pro141 defines a domain responsible for self-association interactions, and that is important for cooperative DNA binding [Yang et al. (1999) Biochemistry 38, 465-477]. We further suggested that the genetically defined gpA-interactive domain in the C-terminal half of the protein is limited to the C-terminal approximately 40 amino acids of gpNu1. Here we describe the cloning, expression, and biochemical characterization of gpNu1DeltaP141, a deletion mutant of gpNu1 that comprises the DNA binding domain and the putative hydrophobic self-assembly domain of the full-length protein. Purified gpNu1DeltaP141 shows a strong tendency to aggregate in solution; However, the protein remains soluble in 0.4 M guanidine hydrochloride, and circular dichroism (CD) and fluorescence spectroscopic studies demonstrate that the protein is folded under these conditions. Moreover, CD spectroscopy and thermally induced unfolding studies suggest that the DNA binding domain and the self-association domain represent independent folding domains of gpNu1DeltaP141. The mutant protein interacts weakly with the gpA subunit, but does not form a catalytically competent holoenzyme complex, suggesting that the C-terminal 40 residues are important for appropriate subunit interactions. Importantly, gpNu1DeltaP141 binds DNA tightly, but with less specificity than does full-length protein, and the data suggest that the C-terminal residues are further required for specific DNA binding activity. The implications of these results in the assembly of a functional holoenzyme complex are discussed.

Tyrosine, phenylalanine, and disulfide contributions to the circular dichroism of proteins: circular dichroism spectra of wild-type and mutant bovine pancreatic trypsin inhibitor.

Improved descriptions of the lowest energy excited states of tyrosine and phenylalanine side chains have been developed in order to extend the capabilities of calculating the circular dichroism (CD) spectra of proteins. Four transitions (Lb, La, Bb, and Ba) for each of the side-chain chromophores were considered, and the transition monopole charges were obtained from a CNDO/S calculation on models representing the individual groups. Monopole charges at midpoints of the bonds, corresponding to the maximum transition charge densities in the Lb band, and monopole charges representing the vibronic coupling with the B transitions for the La transition were also included. The aromatic transitions were combined with the peptide transitions (npi, pi0pi n'pi, and pi+pi) and disulfide transitions (n1sigma and n4sigma) in the framework of the origin-independent matrix method to compute the CD spectra of different crystal forms and Y --> L and F --> L mutants of bovine pancreatic trypsin inhibitor (BPTI). The structures of the mutants were obtained by replacing the appropriate tyrosine or phenylalanine residue by leucine in the wild-type crystal structure. The CD calculations were performed on the energy-minimized structures. The CD spectrum calculated for the form II crystal structure of BPTI showed the best agreement with experiment. In the far UV, the calculated and experimental CD spectra agree to various extents for the wild-type and mutant BPTI. Among the mutants, the calculated CD spectra of Y4L, Y10L, Y23L, and F45L showed reasonable agreement with experiment, while those of Y21L and F22L, the two residues interacting with most aromatic groups, showed poor agreement. In the near UV, the negative bands predicted for the wild-type and mutant BPTI have much less intensity than observed experimentally.

Binding of pyridoxal 5'-phosphate to the heme protein human cystathionine beta-synthase.

Cystathionine beta-synthase (CBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme, catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS was recently shown to be a heme protein. While the role of heme in CBS is unknown, catalysis by CBS can be explained solely by participation of PLP in the reaction mechanism. In this study, treatment of CBS with sodium borohydride selectively reduced the Schiff base but did not affect the heme. Purification and sequencing of the PLP-cross-linked peptide from a trypsin digest of the reduced enzyme revealed the evolutionarily conserved Lys119 to be the residue forming the Schiff base. Serine and hydroxylamine form an alpha-aminoacrylate and an oxime with PLP in CBS, respectively. The sulfhydryl-containing substrate, homocysteine, disturbs the heme environment but does not interact with PLP. In contrast to other PLP-dependent enzymes, CBS emits no PLP-related fluorescence when excited at 296 or 330 nm. PLP but not heme dissociates from the enzyme in the presence of hydroxylamine. The dissociation of PLP is a multistage process involving a short approximately 500 s lag phase, followed by a rapid inactivation and a slower PLP-oxime formation. PLP-free CBS exhibits a decrease of secondary structure as well as loss of CBS activity that can be only partially restored by PLP. This study constitutes the first comprehensive investigation of PLP interaction with a heme protein.

In vitro release kinetics of gentamycin from a sodium hyaluronate gel delivery system suitable for the treatment of peripheral vestibular disease.

For certain patients who experience intense vertigo arising from unilateral vestibular lesions, the primary therapy is a vestibular nerve section, an intracranial surgical procedure. One alternative to this treatment is therapeutic ablation of vestibular function on the unaffected side using an ototoxic agent. We prepared a biodegradable sustained-release gel delivery system using sodium hyaluronate that can be administered into the middle ear using only a local anesthetic. The gel contains gentamycin sulfate, the ototoxic agent of choice for treatment of unilateral vestibulopathy, and it exhibits diffusion-controlled release of the drug over a period of hours. The released gentamycin could then diffuse into the inner ear through the round membrane. This represents an important advance over previous formulations, which used only gentamycin sulfate solutions, in that it should allow more careful control of the dose, it should reduce loss of the drug from the middle ear site, and it should maintain intimate contact with the round membrane. By carefully controlling the dose, it should be possible to inhibit vestibular function while minimizing hearing loss. Herein we describe the in vitro release kinetics of gentamycin sulfate from sodium hyaluronate gels and find that the system obeys Fickian behavior.

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein.

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.

Secondary structure of antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis.

Antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis are among the most active antifreeze proteins known. The Dendroides AFPs (DAFPs) consist of 6 or 7, 12- or 13-mer repeat units with a consensus sequence of -C-T-X3-S-X5-X6-C-X8-X9-A-X11-T-X13-. Nearly all of the Cys residues are in internal disulfide bridges between positions 1 and 7 within the repeats. The study presented here identified the secondary structure of the DAFPs using infrared and circular dichroism (CD) spectroscopies. The eight disulfide bridges impose significant constraints on potential secondary structural features (i.e., a number of three-residue gamma-turns) which may lead to unusual infrared and CD spectra that require special interpretation. At 25 degreesC the DAFPs contain approximately 46% beta-sheet, 39% turn, 2% helix, and 13% random structure. In the presence of ice there is a slight increase in helix and beta-sheet structures and a decrease in both turn and especially random structures. This change in the presence of ice may reflect a certain amount of flexibility in the DAFP structure. These structural changes may permit an improved lattice match between the DAFPs and ice, a requisite for the noncolligative freezing-point-depressing activity of the DAFPs.

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition.

Limited stability impedes the development of industrial and pharmaceutical proteins. Dried formulations are theoretically more stable, but the drying process itself causes structural damage leading to loss of activity after rehydration. Lyophilization is the most common method used to dry proteins, but involves freezing and dehydration, which are both damaging to protein. We compared an air-drying method to freeze-drying to test the hypothesis that terminal dehydration is the critical stress leading to loss of activity. The secondary structure of air-dried and freeze-dried actin was analyzed by infrared spectroscopy and related to the level of activity recovered from the rehydrated samples. Actin dried by either method in the absence of stabilizers was highly unfolded and the capacity to polymerize was lost upon rehydration. The degree of unfolding was reduced by air-drying or freeze-drying actin with sucrose, and the level of activity recovered upon rehydration increased. The addition of dextran to sucrose improved the recovery of activity from freeze-dried, but not air-dried samples. Dextran alone failed to protect the structure and function of actin dried by either method, indicating that proteins are not protected from dehydration-induced damage by formation of a glassy matrix. In some cases, recovered activity did not correlate directly with the level of structural protection conferred by a particular additive. This result suggests that secondary structural protection during drying is a necessary but not sufficient condition for the recovery of activity from a dried protein after rehydration.

Aggregation of recombinant human interferon gamma: kinetics and structural transitions.

Protein aggregation is a complex phenomenon that can occur in vitro and in vivo, usually resulting in the loss of the protein's biological activity. While many aggregation studies focus on a mechanism due to a specific stress, this study focuses on the general nature of aggregation. Recombinant human interferon-gamma (rhIFN-gamma) provides an ideal model for studying protein aggregation, as it has a tendency to aggregate under mild denaturing stresses (low denaturant concentration, temperature below the Tm, and below pH 5). All of the aggregates induced by these stresses have a similar structure (high in intermolecular beta-sheet content and a large loss of alpha-helix) as determined by infrared and circular dichroism spectroscopy. Thermally induced and denaturant-induced aggregation processes follow first-order kinetics under the conditions of this study. Spectroscopic and kinetic data suggest that rhIFN-gamma aggregates through an intermediate form possessing a large amount of residual secondary structure. In contrast to the aggregates formed under denaturing stresses, the salted-out protein has a remarkably nativelike secondary structure.