Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study.

The structural domains of the Escherichia coli CheA protein resemble 'beads on a string', since the N-terminal phosphate-accepting (P) domain is joined to the CheY/CheB-binding (B) domain through a flexible linker, and the B domain is in turn joined to the C-terminal dimerization/catalytic/regulatory domains by a second intervening linker. Dimerization occurs primarily via interactions between two dimerization domains, which is required for CheA trans-autophosphorylation. In this study, sedimentation equilibrium was used to demonstrate significant subunit interactions at secondary sites in the two naturally occurring (full-length and short) forms of CheA (CheA(1-654) or CheA(L), and CheA(98-654) or CheA(S)) by contrasting the dimerization of CheA(L) and CheA(S) to CheA(T), an engineered form that lacked the P domain entirely. The estimated dimer dissociation constant (K(1,2)) for CheA(T) (3.1 microM) was weaker than K(1,2) for CheA(L) (0.49 microM), which was attributed to the P domain-catalytic domain interactions that were present in CheA(L) but not CheA(T). In contrast, CheA(S) dimerization was unexpectedly stronger (K(1,2) approximately 20 nM), which arose through interactions between two P domain remnants in the CheA(S) dimer. This conclusion was supported by the results of sedimentation equilibrium experiments conducted with P domains and P domain remnants expressed in the absence of the dimerization/catalytic/regulatory domains. The P domain remnant had a measurable tendency to self-associate; the full-length P domain did not. Hydrophobic forces probably drive this interaction, since hydrophobic amino acids buried in the intact P domain are solvent-exposed in CheA(S). Also, the nascent N-terminus of CheA(S) bound to the phosphatase (CheZ) more effectively, a conclusion based on the demonstrably greater ability of the P domain remnant to co-sediment CheZ, compared to the intact P domain.

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.

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.

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

The crystallographic structure of the Escherichia coli OXA-1 beta-lactamase has been established at 1.5-A resolution and refined to R = 0.18. The 28.2-kD oxacillinase is a class D serine beta-lactamase that is especially active against the penicillin-type beta-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 Omega loop near the beta-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 Ogamma 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 A 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.

Sedimentation velocity, multi-speed method for analyzing polydisperse solutions.

A method is described for the sedimentation velocity analysis of solutions composed of macromolecular solutes of widely disparate size. In sedimentation velocity experiments, usually a single rotor speed is chosen for the entire run, and consequently, the range of observable sedimentation coefficients can be severely limited. This limitation can be removed if the speed is varied during the run, starting with a relatively low speed so that the largest particles can be easily observed. The speed is increased during the run until full speed is attained and the run continued at full speed until the smallest species of interest have cleared the solution. The method, called wide distribution analysis, is based on the method developed originally by Yphantis and co-workers (Proc. Natl. Acad. Sci. USA 78 (1981) 1431) and on the time derivative method of Stafford (Anal. Biochem. 203 (1992) 295), essentially eliminating both the time-independent and radially-independent noise thereby improving the precision, especially for interference optics. An algorithm for analysis of data from both absorbance and interference optics and experimental protocols compatible with the Beckman XL-I Analytical Ultracentrifuge are presented. With these protocols an extremely wide range of sedimentation coefficients from approximately 1.0 to 250000 S can be accommodated in a single multi-speed run.

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.