Emory H. Braswell

Comparison of β-lactamases of classes A and D : 1.5-Å crystallographic structure of the class D OXA-1 oxacillinase

The crystallographic structure of the Escherichia coli OXA‐1 β‐lactamase has been established at 1.5‐Å resolution and refined to R = 0.18. The 28.2‐kD oxacillinase is a class D serine β‐lactamase that is especially active against the penicillin‐type β‐lactams oxacillin and cloxacillin. In contrast to the structures of OXA‐2, OXA‐10, and OXA‐13 belonging to other subclasses, the OXA‐1 molecule is monomeric rather than dimeric and represents the subclass characterized by an enlarged Ω loop near the β‐lactam binding site. The 6‐residue hydrophilic insertion in this loop cannot interact directly with substrates and, instead, projects into solvent. In this structure at pH 7.5, carboxylation of the conserved Lys 70 in the catalytic site is observed. One oxygen atom of the carboxylate group is hydrogen bonded to Ser 120 and Trp 160. The other oxygen atom is more exposed and hydrogen bonded to the Oγ of the reactive Ser 67. In the overlay of the class D and class A binding sites, the carboxylate group is displaced ca. 2.6 Å from the carboxylate group of Glu 166 of class A enzymes. However, each group is equidistant from the site of the water molecule expected to function in hydrolysis, and which could be activated by the carboxylate group of Lys 70. In this ligand‐free OXA‐1 structure, no water molecule is seen in this site, so the water molecule must enter after formation of the acyl‐Ser 67 intermediate.

Measuring Protein‐Protein Interactions by Equilibrium Sedimentation

This unit describes basic principles and practice of sedimentation equilibrium analytical ultracentrifugation for the study of reversible protein interactions, such as the characterization of self‐association, heterogeneous association, and binding stoichiometry, as well as the determination of association constants. Advanced tools such as mass conservation analysis, multiwavelength analysis, and global analysis are introduced and discussed in the context of the experimental design. A detailed protocol guiding the investigator through the experimental steps and the data analysis is available as an internet resource. Curr. Protoc. Immunol. 79:18.8.1‐8.18.28.

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein.

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self‐association properties of MinE by analytical ultracentrifugation and by studies of hetero‐oligomer formation in non‐denaturing polyacrylamide gels. The self‐association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C‐terminal MinE22–88 fragment forms hetero‐oligomers with MinE+ when the proteins are co‐expressed. In contrast, the MinE36–88 fragment does not form MinE+/MinE36–88 hetero‐oligomers, although MinE36–88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co‐expressed with MinE+ in wild‐type cells. Therefore, hetero‐oligomer formation is not necessary for the induction of minicelling by expression of MinE36–88 in wild‐type cells. The interference with normal septal placement is ascribed to competition between MinE36–88 and the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Molecular Shape, Dissociation, and Oxygen Binding of the Dodecamer Subunit of Lumbricus terrestris Hemoglobin

Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 ± 0.01 nm, maximum diameter = 10.59 ± 0.01 nm, and volume = 255 ± 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1°C and 4 at 25°C, providing ΔH = −20 kcal/mol and ΔS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]−8. Equilibrium oxygen binding measurements at pH 6.5-8.5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9.5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25°C. P50 was decreased 3- and 2-fold in ~100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.

Heparin: Molecular weight and degradation studies☆

Abstract 1. 1. Previously published molecular weights of heparin determined by sedimentation have been found to be incorrect and are probably too low by about 16%. By means of equilibrium sedimentation, the study of molecular weight heterogeneity and the determination both Z- and weight-average molecular weights was made. Heparin was found to be quite heterogeneous with an average ratio of Z-average to weight-average molecular weight of about 1.8. 2. 2. Studies of the physical and chemical properties of heparin degraded by acid hydrolysis so that about half the anticoagulant activity is removed, lead to the conclusion that in the initial phases of this hydrolysis the formation of internal esters may occur. 3. 3. Similar studies performed on a heparin sample submitted to periodate oxidation to a degree of degradation similar to that of the hydrolysis degradation, reveal that more drastic changes occur in this process. These changes involve: the loss of N; oxidative cleavage; and the reduction of molecular weight.

The Effect of pH and Temperature on the Self-Association of Recombinant Human Interleukin-2 as Studied by Equilibrium Sedimentation

The self-association of recombinant human interleukin-2 (rhIL-2) in solution was investigated as a function of pH and temperature using equilibrium sedimentation. Studies were performed at pH 3.6, 6.5 and 8.2, at 1°C and 20°C. A model assuming an ideal single molecular species describes the data observed at pH 6.5 at both temperatures. At pH 8.2, the data from both temperatures can be better described by a weak monomer-dimer association equilibrium. The values of the association constants obtained indicate the presence of less than 10% dimer at a concentration of 1 mg/ml at both temperatures. At pH 3.6, aggregates with a Z average molecular weight of over 35 times that of monomeric rhIL-2 were formed. The smallest associating species present under these conditions corresponds to the monomer, which produces aggregates with a wide range of molecular weights. The monomer appears to be in equilibrium with the smallest aggregates, in that a model describing an indefinite association fits the data obtained at the highest centrifugal speed. No model was found to successfully describe the association of the monomer into the much larger aggregates observed at lower speeds. This may be the result of the lack of rapid thermodynamic reversibility of the larger aggregates. Temperature was found to have no significant effect on the largest aggregates that were formed at pH 3.6.

Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL

Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate.

Quaternary structure of the neuronal protein NAP-22 in aqueous solution

NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is approximately 1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is approximately 0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.

Polyelectrolyte Charge Corrected Molecular Weight and Effective Charge by Sedimentation

Ionic charge on a macromolecule complicates the determination of its molecular weight in solution due to the Donnan effect. Compensation for it can be made if one knows the value of the effective charge, which can be found by dialysis equilibrium across a semipermeable membrane. A moving boundary of molecules sedimenting in a centrifugal field can act as a membrane, obviating some of the disadvantages (such as selective adsorption) of a real membrane. Interference optics are used to monitor the reverse gradient of the salt due to the Donnan effect, hence facilitating the determination of the effective charge. The apparent molecular weight obtained from a conventional sedimentation equilibrium can then be corrected to yield the true molecular weight. The effective charge is valuable in revealing macromolecular structural features when related to the titratable charge through the Manning counter-ion condensation theory. Agreement between the values of the backbone molecular weights for the Na, Cs, and Ca salts of heparin indicated the validity of the approach. The effective charge ratio and the axial charge spacing for the Na and Ca heparin agreed with the literature, whereas the results for Cs indicated a degree of binding in excess of that due to counter-ion condensation.

Analysis of factors affecting lactic dehydrogenase subunit composition determinations

Abstract The ratios of the activities exhibited at low and high pyruvate levels, here designated as R values, for prepared mixtures of parental LDH tetramers will not fall on a straight line in a plot of R versus per cent A or B subunits (by activity) unless either the R value for the A4B0 tetramer (RA) or the R value for the A0B4 tetramer (RB) is equal to one. Intratetrameric catalytic independence is not necessary for such linearity. R values for the five electrophoretically resolved LDH tetramers will not fall on a straight line in a plot of R versus per cent A or B subunits (denoting per cent concentration of the subunits) unless the turnover number of the A subunits at the high substrate level (TAH) is equal to the turnover number of the B subunits at the high substrate level (TBH) for R, or the turnover number of the A subunits at the low substrate level (TAL) is equal to the turnover number of the B subunits at the low substrate level (TBL) for 1/R, and there is intratetrameric catalytic independence. Per cent A or B subunits by activity (f) is not equatable with per cent A or B subunits by subunit concentration (F) unless TAH is equal to TBL, and there is intratetrameric catalytic independence, i.e. the two standard curves will not be the same. The study of one set of data on chicken LDH isozymes indicates that conditions are not met under which f and F will be equivalent, or under which plots for their equations will be linear. This observation leads to the interesting conclusion that concomitant with the formation of hybrid tetramers there may be an alteration of the catalytic properties of at least the A-type subunits. The determination of turnover numbers of all the isozymes of a given series at both conditions of the differential assay is, therefore, necessary to the establishment of an assay procedure for determining the mole fraction of a given subunit present in an isozyme mixture. This conclusion infers the approximate nature of previous differential assay procedures, and removes the need for linearity of R values in such methods. Intratetrameric catalytic dependence is interesting both in terms of enzyme subunit interaction and its metabolic consequences.