Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins

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.     

Functional analysis of the domains in Cox11

Cox11 is an intrinsic mitochondrial membrane protein essential for the assembly of an active cytochrome c oxidase complex. Cox11 is tethered to the mitochondrial inner membrane by a single transmembrane helix. Domain mapping was carried out to determine the functional segments of the Cox11 protein. The C-terminal 189 residue Cu(I)-binding domain is shown to be exposed within the mitochondrial intermembrane space. This orientation was demonstrated by the proteolytic susceptibility of a C-terminal Myc epitope tag in mitoplasts but not intact mitochondria. Fusion of the N terminus of Cox11 to the matrix ribosomal protein Rsm22 results in a functional protein capable of suppressing the respiratory defect of both Deltacox11 cells and Deltarsm22 cells. The functionality of the fusion protein suggests that the Cox11 N terminus projects into the matrix. The fusion of the C-terminal segment of Cox11 to Rsm22 resembles a naturally occurring fusion of Cox11 in Schizosaccharomyces pombe to a sequence homologous to the Saccharomyces cerevisiae Rsm22. Studies on a series of SCO1/COX11 chimeras reveal that the matrix domain of Cox11 lacks a specific function, whereas the Cu(I) binding/donating function requires the yeast Cox11 sequence. The Cu(I)-binding domain from human Cox11 cannot functionally replace the yeast sequence. The copper domain of Cox11 may be an important docking motif for Cox1 or a Cox1-associated protein.     

The repair of potentially lethal damage and sublethal damage in strains of mouse L5178Y lymphoma cells differing in radiation sensitivity

Mouse lymphoma strains L5178Y-R (LY-R) and L5178Y-S (LY-S), which are differentially sensitive to the cytotoxic effects of ionizing radiation, were found to differ in their abilities to repair potentially lethal damage (PLD) and sublethal damage (SLD). The results showed that strain LY-R was more proficient than strain LY-S in the repair of SLD. The split dose recovery observed in strain LY-S could be accounted for by its recovery during postirradiation incubation. In contrast, SLD repair occurred in the absence of PLD repair in strain LY-R. The possibility that the repair of PLD might be completed prior to the postirradiation incubation in strain LY-R was suggested by the decreased survival observed when the cells were irradiated in a hypotonic solution. The repair of PLD and SLD in strain LY-S was temperature sensitive, occurring during postirradiation incubations between 15 and 34 degrees C, but not at 37 or 40 degrees C. This temperature sensitivity is very similar to the temperature sensitivity of the repair of pH 9.6-labile lesions in DNA in strain LY-S, as reported previously. Thus postirradiation cellular recovery processes in strain LY-S may involve the repair of pH 9.6-labile lesions in DNA. Temperature-dependent changes in the postirradiation distribution of cells throughout the cell cycle were observed which could contribute to the temperature sensitivity of the postirradiation recovery of strain LY-S.     

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.     

Heavy chain variable region allotypic subspecificities of rabbit immunoglobulins. II. Selective escape of the a1-AB Ig subpopulation after the induction of auto anti-a1 antibody.

An a1a2 rabbit (P286-3), neonatally suppressed for the expression of the a1 allotype, was immunized with autologous a1 IgG at 2 months of age. Both auto anti-a1 Ab and a1 IgG molecules were found in the serum of this rabbit after the auto-immunization. The auto anti-a1 Ab and the IgG from the auto anti-a1 Ab-depleted serum were isolated. Of the previously defined a1-AB, a1-AC, and a1-AD Ig subpopulations, the a1 IgG in the IgG preparation from the rabbit P286-3 were all of the a1-AB Ig subpopulation. The auto anti-a1 Ab from rabbit P286-3 did not react with the a1-A, a1-B, and a1-C allotypic subspecificities; thus, it was presumably specific for the a1-AC and a1-D allotypic subspecificity. Thus, the a1-AB Ig subpopulation escaped from allotype suppression in rabbit P286-3, whereas the a1-AD Ig subpopulation remained suppressed. The a1-AD Ig subpopulation will probably remain suppressed for a long time and perhaps permanently since rabbit P286-3 has produced circulating auto-Ab specific for the a1-D allotypic subspecificity. These results indicate that the a1 Ig subpopulations are synthesized by distinct clones of lymphocytes under separate control.     

A selective inhibitor of serotonin uptake: Lilly 110140, 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine

Lilly 110140 is a highly selective inhibitor of serotonin uptake into synaptosomes of rat brain, unlike previous monoamine uptake inhibitors that also inhibit the uptake of norepinephrine and dopamine. Lilly 110140 should be useful in studying the function of serotoninergic neurons and may be helpful in clariying the role of serotonin in certain types of mental depression.     

Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria

A23187, a new antibiotic with ionophore properties, uncoupled oxidative phosphorylation in mitochondria which oxidized either malate plus glutamate or succinate. Ca²⁺, but not Mg²⁺, enhanced the uncoupling effect. Fluorescence of ANS¹ was increased by A23187 suggesting the mitochondrial membranes were de-energized. This de-energization was presumably by activation of the energy-dependent uptake of Ca²⁺. The steady-state measurements of murexide-divalent cation complexes showed that A23187 caused mitochondria to release the accumulated Ca²⁺ to the medium. This reduced the transmembrane Ca²⁺ gradient even though normal active Ca²⁺ uptake could take place. A23187 inhibited activity of ATPase induced by 2,4-dinitrophenol, valinomycin, and Ca²⁺. The addition of Mg²⁺ could prevent this inhibition presumably by maintaining the endogenous Mg²⁺ concentration. The above metabolic events could be explained by the fact that molecules of A23187 function in the mitochondrial inner membrane as mobile carriers for divalent cations.     

Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine

A recent analysis of amino acid sequence data (Graur et al.) suggested that the mammalian order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue is whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. To resolve this problem, we sequenced a region (2,645 bp) of the mitochondrial genome of the guinea pig containing the complete 12S ribosomal RNA, 16S ribosomal RNA, and transfer RNA(VAL) genes for comparison with the available sciurognath and other mammalian sequences. Several methods of analysis and statistical tests of the data all show strong support for rodent monophyly (91%-98% bootstrap probability, or BP). Calibration with the mammalian fossil record suggests a Cretaceous date (107 mya) for the divergence of sciurognaths and hystricognaths. An older date (38 mya) for the controversial Mus- Rattus divergence also is supported by these data. Our neighbor-joining analyses of all available sequence data (25 genes) confirm that some individual genes support rodent polyphyly but that tandem analysis of all data does not. We propose that the conflicting results are due to several compounding factors. The unique biochemical properties of some hystricognath metabolic proteins, largely responsible for generating this controversy, may have a single explanation: a cascade effect resulting from inactivation of the zinc-binding abilities of insulin. After excluding six genes possibly affected by insulin inactivation, analyses of all available sequence data (7,117 nucleotide sites, 3,099 amino acid sites) resulted in strong support for rodent monophyly (94% BP for DNA sequences, 90% for protein sequences), which lends support to the insulin-cascade hypothesis.