(Received for publication, October 10, 1995; and in revised form, January 5, 1996)
From the
To study the role of omega loop D, residues 70-84, in the
structure and function of yeast iso-1-cytochrome c, this loop
was replaced with homologous and heterologous loops. A novel method was
developed for rapid insertion of these mutations into the yeast
chromosome at the CYC1 locus. The strains containing these
loop replacement cytochromes cannot grow on nonfermentable carbon
sources, indicating that the proteins are nonfunctional. Whole cell
difference spectroscopy shows that no holocytochrome c is
present; however, apoprotein is found by immunoblot analysis. Thus,
apoprotein is present in these mutant strains, but it cannot bind heme
and cannot compete with wild type apoprotein conversion to holoprotein.
This is a unique example of a set of loop replacements that do not
produce folded protein, and these results suggest that the loop D amino
acid sequence in iso-1-cytochrome c plays a significant role
in cytochrome c biosynthesis in vivo. To identify the
significant amino acids in loop D, random mutagenesis of six highly
conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and
Lys-79, was accomplished. Sequencing of the random mutants shows that
strict conservation of none of these residues is required to produce a
minimally functional cytochrome c. Preferences are found for
small, hydrophilic or aromatic residues at position 74, hydrophobic
residues at position 75, glycine and arginine at positions 76 and 77,
and -branched amino acids at position 78. Implications for the
role of loop D in the structure and function of iso-1-cytochrome c are discussed.
The protein folding problem is an important, unsolved problem in
molecular biology. An understanding of this problem is important to
utilization of the data being generated by the human genome project and
to further the design and engineering of proteins with specific
structures and functions. Loops and turns, the connections between the
regular secondary structures, are critical to the design of properly
folded proteins. These structures are difficult to characterize because
of their range of conformations. The short -turns and reverse
turns were the first to be characterized(1, 2) ; then
the longer
-loops were described(3) ; and finally a more
comprehensive definition of loops was published ((4) ; for
review, see (5) ). We are interested in defining the role that
loops, particularly
-loops, play in protein folding, function,
and stability.
-Loops are nonregular protein secondary
structures defined as a sequence of six or more residues with a short
distance between segment termini and a lack of regular hydrogen bonding
between backbone residues(3) . However, significant hydrogen
bonding and hydrophobic interactions can exist between side chain and
backbone atoms within the loop itself. (
)
-Loops are
generally found at protein surfaces(3) . Because they are
positioned at protein surfaces, changes in the loop amino acid
sequence, as well as changes in loop length, are generally less
disruptive to the rest of the protein than changes made in residues in
the protein core. Indeed, previous studies have found that large
changes within loop regions rarely affect the ability of the protein to
fold, although the function of the protein may be disrupted if the loop
plays an important role in protein function (for review, see (5) ). Thus, loop replacement or loop swap experiments are good
methods with which to analyze the role that a loop plays in protein
function; furthermore, the importance of loops for protein folding and
stability can be determined by spectroscopic and crystallographic
studies of loop replacement proteins(6, 7) .
The
significance of -loop D, residues 70-84, to the structure
and function of iso-1-cytochrome c of Saccharomyces
cerevisiae is investigated here. Cytochrome c is a small
protein found in the electron transport system of most organisms.
Iso-1-cytochrome c shuttles electrons from cytochrome c reductase to cytochrome c oxidase within the
inter-mitochondrial space(8, 9) . It also interacts
with cytochrome c peroxidase, cytochrome b
, and sulfite oxidase. Despite sequence
variation, a general tertiary fold is found in c-type
cytochromes from prokaryotic and eukaryotic
organisms(8, 10) . The overall tertiary fold consists
of three
-helices, one at the amino terminus, one at the carboxyl
terminus, and one near residues 60-64, and three or four
-loops, surrounding a heme group that is covalently bound to the
protein by two thioether linkages to cysteine
residues(8, 10) . The positions of the
-loops
are conserved, although the loops are the major sites of insertions and
deletions between family members from different species(11) .
This general tertiary structure is depicted in Fig. 1.
Figure 1:
Ribbon structure of iso-1-cytochrome c. The -loops and helix secondary structure regions of S. cerevisiae iso-1-cytochrome c(47) are
displayed as various shades of gray and labeled. The secondary
structures are designated as amino-terminal helix (N-helix),
residues 2-11; loop A, residues 18-32; loop B, residues
34-43; loop C, residues 40-54; 60s helix, residues
61-69; loop D, residues 70-84; and carboxyl-terminal helix (C-helix), residues 88-101. The side chains of Tyr-74,
Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79 are shown as ball-and-stick-figures and are
labeled.
The advantage of studying mutations in S. cerevisiae cytochrome c is that mutants that affect the protein structure and those that affect the function can be distinguished in vivo. Because S. cerevisiae requires a functional cytochrome c to grow on nonfermentable carbon sources, function can be assayed by growth of mutant yeast strains on lactate, glycerol, or ethanol media(12) . The presence of holoprotein, cytochrome c to which the heme group is covalently attached, can be detected by whole cell difference spectroscopy following growth of cells in dextrose medium, regardless of the functionality of the protein(12, 13) . A lack of holoprotein would indicate that a mutation has altered the fold or stability of apo- or holoprotein so that it is more readily proteolyzed or that it has disrupted a step in the biosynthetic processing of holocytochrome c, such as transcription in the nucleus, translation on free ribosomes, mitochondrial recognition and import, or recognition by cytochrome c heme lyase, the protein that covalently attaches heme to the apoprotein(14, 15) .
Previous loop
deletion and replacement experiments have been done on -loops in
yeast iso-1-cytochrome c. That work established that deletion
of a portion of loop A, residues 21-28, or loop D, residues
74-78, resulted in a complete deficiency of holocytochrome c protein; however, holoprotein was still produced when portions of
two other loops, loops B and C, were deleted(11) . Replacement
mutations at the loop A position yielded results similar to the loop
replacement experiment results described for other proteins: the mutant
proteins are altered in stability or function yet are still able to
fold into a native-like holoprotein. In each loop A replacement mutant,
a heme-bound native-like cytochrome molecule is found in the cell,
although the protein is not always functional(6, 11) .
In the current study, loop D was the target of loop replacement experiments so that the role of this loop in protein function and structure could be studied. Here we describe a novel, rapid method for insertion of these mutations into the yeast genome at the wild type iso-1-cytochrome c (CYC1) locus. This protocol was developed so that the mutated cytochromes c are under the same regulatory controls as wild type cytochrome c, and only one copy of the mutant gene exists in the haploid yeast cell. Amino acids at the loop D position in yeast iso-1-cytochrome c were replaced by homologous loop residues from prokaryotic c-type cytochromes and by heterologous loops from other proteins. The ability of the mutant proteins to fold and function was tested in vivo. An in vivo competition study was designed to determine whether the mutated apoproteins could compete with wild type holoprotein production. Because none of the loop replacement mutations produced a folded protein, directed, random mutagenesis was applied to six highly conserved residues within loop D: Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79. (Five of these six residues are those that had previously been deleted in the loop D deletion mutation.) The sequence variability that is allowed in these residues while still producing a minimally functional cytochrome c was thus identified. Although random mutagenesis has previously been applied to interior residues and hydrophobic cores, this is the first description of random mutagenesis of a loop region in a protein and is a step toward identifying the importance of loop-loop and loop-protein interactions in the structure, folding, and function of globular proteins.
The genotype of the strains used in this study are as follows: Escherichia coli HB101: supE44, hsdS20 (rBmB), recA13, ara-14, proA2,
lacY1, galK2, rpsL20, xyl-5, mtl-1(16) ; E. coli CJ236: CAM, dut, ung, F`(17) ; S. cerevisiae strain B7684: MATa, cyc1-814::URA3, cyc7-68::cyh2, his3-
1, ura3-52, leu2-3,112,
trp1-289, can1-100, cyh2 (obtained from F. Sherman);
B2111: MAT
, cyc1-239, arg4-77, cyc7(12) (obtained from F. Sherman);
GH2: MAT
, trp1, leu2-3, leu2-112, gal1
, cyc7::LEU2,
his3 (obtained from R. Zitomer); C6 (wild type): MATa, CYC1, LEU2, cyc7-68::cyh2, his3-
1,ura3-52, leu2-3,112, trp1-289, can1-100, cyh2; C15 (del cyc1): MATa,
cyc1, LEU2, cyc7-68::cyh2, his3-
1, ura3-52, leu2-3,112, trp1-289, can1-100, cyh2. Strain C93 is
isogenic with C6 except that it contains the C102T mutation in the CYC1 gene, as described above.
For incorporation of mutant cytochromes c in the CYC1 locus of the yeast genome, the yeast
integration plasmid pYC30 was constructed from plasmid pAB571 (kindly
provided by T. Cardillo). pYC30 contains an origin of replication and
an ampicillin resistance gene for maintenance and selection in E.
coli, a LEU2 marker for selection in yeast, and an f1(IG)
origin of replication for single-stranded DNA production after R408
infection. It also contains the 2,530-bp ()BamHI-EcoRI chromosomal fragment
containing the wild type CYC1 gene, although the 3` EcoRI site was mutated to a BamHI site so that the
plasmid could be easily digested with BamHI before integration
into the yeast chromosome. The 2.2-kilobase pair SalI-XhoI LEU2 fragment was inserted at the BglII site 515 bp from the start of the BamHI-EcoRI CYC1 fragment. The CYC1 fragment is flanked by MluI sites 262 bp upstream and 197
bp downstream of the beginning and end of the CYC1 locus,
respectively. In the plasmid variant, pYC31, the 789-bp MluI
fragment containing CYC1, was removed and the plasmid
religated, so that mutant cyc1 genes could easily be ligated
into this integration plasmid (see Fig. 2).
Figure 2:
Yeast integration plasmid pYC30 and novel
strategy for integration of mutant cyc1 genes into yeast
chromosome at the CYC1 locus. Plasmid pYC30 was created for a
rapid method of integration of cytochrome c mutant genes into
the yeast chromosome. The plasmid carries the 4.9-kilobase pair BamHI-EcoRI fragment from the CYC1 locus.
The second EcoRI site was mutated to a BamHI site.
For selection in yeast, the 2.2-kilobase pair LEU2 fragment
was inserted into the BglII site of this BamHI-BamHI fragment. The plasmid also contains an
origin of replication and ampicillin resistance gene for maintenance
and selection in E. coli. For integration into the yeast
chromosome, variants of plasmid pYC30 containing mutant cyc1 genes were linearized by BamHI digestion, and the
restriction digest mixture was transformed into a haploid yeast strain,
B7684, using standard lithium acetate protocols(21) . Strain
B7684 is URA3 and leu2
. If the BamHI-BamHI
fragment recombines correctly, positive transformants should be ura
and LEU
. Thus,
transformation mixtures were plated on SD medium lacking leucine to
select for integration of the LEU2 gene. Selected
transformants were then tested on SD(-ura) medium to determine
whether integration resulted in the removal of the cyc1::URA3 fragment from the chromosome as predicted.
Note: for clarity, the figure is not drawn to
scale.
Colonies were screened by purification of the plasmid DNA using the alkaline lysis plasmid preparation (20) and then digested with selected restriction enzymes. Fragments were separated and analyzed by agarose or polyacrylamide gel electrophoresis. Correct insertions resulted in an increase in the size of an AvaII band or a creation of a new restriction site. Positive candidates were confirmed by dideoxy sequencing (U. S. Biochemical Corp. Sequenase kit) of NaOH-denatured plasmid DNA prepared by large scale plasmid preparation (Promega).
Yeast cells of strain B7684, in which the CYC1 gene has been replaced with URA3 gene, were made competent by lithium acetate protocol (21) and transformed with pYC31-derived plasmids linearized with BamHI. Transformants were plated on synthetic dextrose (SD) medium (0.67% yeast nitrogen base, without amino acids and ammonium sulfate; 2% dextrose; 2.2% ammonium sulfate) without leucine SD(-leu) to select for integration of the LEU2 marker through homologous recombination. Selected transformants were then screened on SD medium lacking uracil SD(-ura) for removal of the cyc1::URA3 fragment from the chromosome. Several yeast colonies that grew on SD(-leu) but not on SD(-ura) were selected, subcloned on YPD medium (1% yeast extract, 2% peptone, 2% dextrose), restreaked on SD(-ura), SD(-leu), SD(-trp), and SD(-his) to check strain phenotype. The cyc1 locus of one or two of the colonies that tested correctly was sequenced.
An overnight culture was made by inoculating a single colony of the diploid cytochrome c mutant in YPD medium and growing overnight at 30 °C. After growth for 16 h, the cultures were synchronized by placing them at 4 °C overnight. Cells were centrifuged and rinsed with liquid lactate medium. Ten ml of liquid lactate medium in side arm flasks (0.67% yeast nitrogen base, without amino acids and ammonium sulfate; 1% lactate; 2% ammonium sulfate) was inoculated with a quantity of synchronized cells to start the culture at a density of exactly 8 Klett units (KU). Cell density was measured by a Klett-Summerson electric colorimeter. Culture flasks were shaken at 130 rpm at 25, 30, or 37 °C, depending on the experiment, and cell density was measured at 3-4-h intervals over a 25-30-h period. Yeast strains C6 (wild type), C93 (wild type, C102T) and C15 (del cyc1) are isogenic with the experimental strains, except at the iso-1-cytochrome c locus. C6 and C93 were included as positive controls, and C15 was included as a negative control.
For Western blots, proteins were transferred to 0.45-µm nitrocellulose sheets (Bio-Rad) and washed in blocking buffer (5% low fat dry milk in PBS-T (80 mM disodium hydrogen phosphate, 20 mM sodium dihydrogen phosphate, 100 mM sodium chloride, and 0.1% Tween 20)). Blots were incubated with anti-cytochrome c rabbit serum, which recognizes both apo- and holocytochrome c (compliments of M. Dumont). Blots were washed with PBS-T and incubated further with anti-rabbit Ig, horseradish peroxidase-linked whole antibody (Amersham Corp.) for 30 min. Further washes with PBS-T were followed by detection with enhanced chemiluminescence reagents (Amersham Corp.). The blots were wrapped immediately in plastic wrap and exposed to autoradiography film (Fuji) for 1-5 min at room temperature.
3R/4R: 5`-GAGTACTTGACGAATCCAAAGAAATATATT NNG/C NNG/C ACCAAGATGGCCTTTGGTGGGTTGAAG-3`.
8R: 5`-GAGTACTTGACTAACCCAAAGAAATATATTCCTGGT NNG/T NNG/T ATGGCCTTCGGTGGTTTGAAG-3`.
9R: 5`-GAGTACTTGACTAACCCAAAGAAA NNG/T NNG/T CCTGGTACCAAGATGGCCTTCGGTGGTTTGAAG-3`.
Thirty µg of each oligonucleotide was transformed into yeast strain C51, which contains a deletion of five residues in loop D and does not produce a folded cytochrome c. Transformed cells were plated on YPD plates and incubated at 25 or 30 °C for 4-8 days. Colonies that grew over the background lawn, indicating that an oligonucleotide had integrated at the CYC1 locus to produce a functional cytochrome c, were picked, subcloned, checked for phenotype on SD(-leu), SD(-ura), and SD(-trp) plates. The CYC1 locus in these strains was then sequenced by PCR amplification of chromosomal DNA, as described above. The entire CYC1 locus of all of these strains was sequenced at least once. To verify sequence accuracy, many were sequenced twice.
S. cerevisiae is
an ideal system for differentiating between the structural and
functional effects of mutations in the cytochrome c protein
because structure and function can be distinguished in vivo;
however, to assay structure and function accurately, only one copy of
the mutant gene must be present in the cell. To ensure that this one
copy is under normal regulatory control, it is necessary to integrate
the mutant gene into the CYC1 locus. For this study, an
efficient system for integration was designed in which the integration
plasmid, pYC30, contained the LEU2 gene upstream of the cyc1 coding region. The integration strategy is illustrated in Fig. 2. In B7648, the yeast integration strain, the CYC1 gene is replaced by URA3 and is thus URA; the strain also contains a mutation at
the LEU2 locus and is thus leu
.
When the BamHI-BamHI fragment from pYC30 containing
the CYC1 gene is integrated properly into this yeast strain,
it becomes ura
and LEU
. This efficient method was utilized for
integration of each cyc1 loop D replacement mutation into the CYC1 locus. Typically, approximately 80% of all yeast
transformants that grow on SD medium lacking leucine do not grow on
SD(-ura) plates and are colonies that have correctly integrated
the mutant gene at the CYC1 locus.
Loop D replacement mutations were created by oligonucleotide-directed mutagenesis of the parent pYC17 (DelD) plasmid, integrated into the yeast chromosome, and confirmed by sequence analysis of a PCR-amplified fragment from the yeast chromosome. Isogenic strains containing wild type cytochrome c (C6: wild type; and C93: wild type, C102T), a complete deletion of cytochrome c (C15, del cyc1), and a partial deletion of loop D, residues 74-78, (C51, DelD) were also created and were used for direct comparison with the mutant strains in the in vivo structural and functional studies. A summary of the loop D sequences of these strains is given in Table 1.
Growth of mutant strains was determined by monitoring the cell
density in liquid lactate for a period of 25-30 h at 30 °C (Fig. 3). These growth curves were compared with those of
control strains, C6 (wild type), C93 (wild type, C102T), C15 (del cyc1), and C51 (DelD). The small increase in the number of KU
at the beginning of the experiment (8 KU) and at the end of monitored
growth (20 KU) observed with the cytochrome c deletion
strain may be caused by residual dextrose in the culture medium from
the original synchronized culture, by cell lysis, or by evaporation of
medium due to aeration and not as an ability to utilize lactate as a
carbon source. Mutant yeast strains that display growth comparable to
the cytochrome c deletion yeast strain are interpreted as
having a nonfunctional cytochrome c. As reported previously,
DelD was unable to grow on lactate(11) , and placing this
mutation in our background strain produces the same results (Fig. 3). All of the loop D replacement mutations did not grow
on lactate, indicating that all mutant strains were deficient in a functional cytochrome c. To determine if the mutant
proteins were unstable and sensitive to temperature, growth curves were
also performed at 25 °C; no function was observed at this
temperature either (Fig. 3).
Figure 3: In vivo growth curves in lactate medium. Growth of diploid mutant yeast strains in liquid lactate medium is monitored to determine if the loop D replacement cytochromes c are functional. Growth curves were performed at 25 °C (panel A) and 30 °C (panel B). Log of cell density (KU, measured on a Klett-Summerson colorimeter) is plotted against time of culture. Growth of mutant strains were compared with isogenic strains that have wild type (C102T) cytochrome c (C93) and which are deficient in cytochrome c (C15).
To assay for a decrease in the holoprotein levels or a decrease in the function of the protein to a level that is 1% of normal cytochrome c(26) , cells were plated on glycerol medium. None of the loop D mutant yeast strains was able to grow on glycerol plates, indicating that not even low levels of functional holoproteins are present (data not shown). Possible causes for these results are that no holoprotein is produced, or that holoprotein is produced, but is nonfunctional, or that holoprotein is extremely unstable and is not detectable by these assays.
Figure 4: In vivo low temperature difference spectroscopy. Low temperature whole cell spectroscopy was used to determine if the mutant loop D proteins could enter the mitochondria and bind a heme group. Diploid mutant yeast strains were grown in YPD medium for 15-16 h at 25 °C (panel A) and 30 °C (panel B). To control for the number of cells present in the sample, cell density was measured on a Klett-Summerson colorimeter by diluting the cells 1:40 or 1:200 with 0.1 M sodium phosphate buffer, pH 7.0. 250-µl samples, made from the stock cells, gave a density of 350 KU based on the cell density and dilution factor of the diluted cells. One 250-µl sample of cells (350 KU) was reduced, and another 250-µl sample (350 KU) of the cells was oxidized. Each sample was then mixed with 250 µl of saturated sucrose. Cells were frozen in liquid nitrogen, and a difference spectrum was recorded from 500 to 650 nm. The 548 nm peak characteristic of cytochrome c at low temperatures was monitored for each replacement mutant. Spectra from 540 to 575 nm are shown to highlight this region. Yeast strains C93 (wild type, C102T) and C15 (del cyc1) were monitored as positive and negative controls representing 100 and 0% cytochrome c, respectively.
Figure 5: Immunoblot analysis. Equal volumes of whole cell protein extracts from yeast strains (22) with the same culture density were run on a three-phase SDS-Tricine polyacrylamide denaturing gel (23) and transferred to nitrocellulose. The primary antibody recognizes both apo- and holocytochrome c. The ECL rabbit peroxidase secondary antibody was used, and blots were immersed in ECL reagents before exposing the film with the blots. The apoprotein present in the loop D deletion and replacement mutants were determined by comparing with cytochrome c-deficient yeast cell protein lysates. In addition, the bands were compared with purified apo- (apo lane) and holocytochrome c (holo lane) and to SDS-17S molecular weight marker (Sigma). Protein lysates from mutant yeast stains are labeled as designated in Table 1.
The codon sequences shown in Table 2, Table 3, and Table 4and summarized in Table 5show that individual codons do not appear to be overwhelmingly favored. For example, arginine is encoded 12 times by AGG and nine times by CGG. Leucine is found 15 times in the data base; 7 times it is encoded by CTG, 4 times by TTG. Arginine codons CGT and CGC and leucine codons CTT and CTG cannot be compared because they were encoded only in one or the other of the oligonucleotides. Thus, overwhelming codon preferences, which would result in apparent amino acid preferences, do not appear to occur with our random mutagenesis protocol. Because of the number of mutants, small, statistically significant codon preferences among the oligonucleotides cannot be ruled out.
Twenty-eight colonies obtained from random mutagenesis of Tyr-74 and Ile-75 with oligonucleotide 9R were sequenced, and the data are shown in Table 2, Table 5, and Table 6. The wild type sequence did not appear in any of these 28 strains, demonstrating that we have not sampled all possible functional sequences. There are five sequences that appear in more than one of the mutant strains sequenced: Arg-74 and Val-75 in 9R-18, 23 and 31, Ser-74 and Leu-75 in strains 9R-7 and 19; Trp-74 and Ala-75 in 9R-3 and 25; Gly-74 and Val-75 in 9R-5 and 24; and Met-74 and Leu-75 in 9R-2 and 20. There is a prevalence of bulky residues, particularly tryptophan, and small residues (serine, threonine, and glycine) at position 74. With two exceptions, histidine and glutamine, only hydrophobic residues are found at residue 75 among the 9R mutant strains.
Of the 195 colonies obtained from random mutagenesis of Pro-76 and Gly-77, the CYC1 locus from the chromosomal DNA of 47 colonies was sequenced. The results are shown in Table 2and summarized in Table 5and Table 6. One of the colonies produced using the 3R and 4R oligonucleotides, 3R-8, returned a wild type sequence. There are several sequence combinations that appear in more than one of the mutant strains sequenced: Gly-76 and Gly-77 in strains 3R-11, 3R-25, and 4R-11; Gly-76 and Arg-77 in strains 3R-10 and 3R-24; Gly-76 and Phe-77 in strains 3R-2 and 4R-43; Arg-76 and Leu-77 in strains 3R-16 and 3R-18; Arg-76 and Gly-77 in strains 3R-5 and 3R-17. This result suggests that an adequate number of mutant strains have been sampled to study the general trends in the Pro-76 and Gly-77 mutants; however, it is probable that other residue combinations that produce a functional cytochrome c would be found if more strains were sequenced.
The data in Table 2and Table 6show that glycine, arginine, and tryptophan are overwhelmingly preferred at position 76, whereas arginine and glycine are found often at position 77. This is a curious result because glycine is the smallest amino acid, whereas tryptophan and arginine are two of the largest. The distribution of amino acids found at these positions does not seem to change as the isolation temperature is changed from 30 to 25 °C (Table 6); however, the number of mutants sequenced thus far is not sufficient to make a statistical argument.
Forty-one colonies obtained from random mutagenesis of
Thr-78 and Lys-79 with oligonucleotide 8R were sequenced (Table 2, Table 5, and Table 6). One colony, 8R-3,
returned a wild type sequence. Several strains returned the same
sequence combinations: valine and serine in 8R-8, 26 and 36; alanine
and serine in 8R-25, 40, and 47; glycine and alanine in 8R-17 and 19;
and serine and arginine in 8R-18 and 23. A preponderance of
-branched residues, isoleucine, threonine and valine, and small
residues, glycine, alanine and serine, are found at position 78. Serine
and arginine appear to be preferred at position 79. Incubation
temperature does not produce a significantly different set of amino
acids at these positions (Table 6).
Although random mutagenesis does not uncover any residues that are strictly conserved and required for biosynthesis or function of holocytochrome c, a number of preferences were uncovered. Because of the random mutagenesis protocol that we used, all of the residue combinations discovered produce a functional, and thus folded, cytochrome c; however, the mutant proteins may be minimally functional. An understanding of the exact effect of these mutations on the structure and function of cytochrome c can now be determined by further examination of structure and function in these mutants.
The importance of the conserved loop D sequences in eukaryotic cytochromes c for function and biosynthesis of this protein has now been examined. Mutant proteins containing loop D replacements were assayed in vivo for function and structure. The chromosomal integration system that we have developed and described here (Fig. 2) allows us to study the effects of loop D replacement mutations inserted at the CYC1 locus in yeast. Moreover, the effects of these mutations can be analyzed at the normal wild type cytochrome c expression levels to discover the actual effect of the mutation on growth of the yeast.
All loop replacement mutants examined here failed to produced holoprotein, so directed random mutagenesis was performed to identify specifically those residues important for the structure, folding, or biosynthesis of cytochrome c. Of the six highly conserved loop D residues that were randomly mutated, none is strictly required for the folding or structure of cytochrome c. Residue preferences can now be examined in more detail.
Multiple
examples of loop deletions and replacements show that such mutations do
not affect the overall structure of the protein, although protein
function and stability are often affected. In iso-1-cytochrome c, loop B, residues 34-45, and loop C, residues
40-54, were deleted, and the resulting proteins folded and were
functional, although less so than the wild type protein(11) . A
six-residue deletion of a mobile loop in Staphylococcal nuclease, located near a putative catalytic site, resulted in a
more active, more stable protein(28) . NMR experiments
demonstrated that this mutation had essentially the same structure as
the wild type protein, except that the larger loop was replaced with a
smaller -turn(28) . A deletion of four residues from the
mobile loop of triose phosphate isomerase resulted in a folded protein
with a significant decrease in specific catalytic activity, suggesting
that this mobile loop has a role in the catalytic reaction
mechanism(29) . The function of a mobile loop in E. coli dihydrofolate reductase was studied by deleting a highly conserved
four-residue region within the center of the loop and sealing it with a
glycine residue(30) . The major effect of this loop deletion
was to decrease loop flexibility. In both dihydrofolate reductase and
triose phosphate isomerase, structural studies indicate the flexibility
of the loop allows conformational changes necessary for performing the
function of stabilizing intermediates along the reaction
pathway(29, 30) .
Loop replacements also generally result in a folded protein, although if the loop is involved in protein function the resulting hybrid protein is not necessarily functional. Replacement of hypervariable loop regions in the immunoglobulins results in folded immunoglobulin proteins with altered antigen specificity(31) . Swapping of variable loops of homologous ribonuclease proteins, RNase and angiogenin, results in an exchange of substrate specificity between the two proteins(32, 33) . Although deletion of loop A, residues 18-32, in cytochrome c resulted in a complete deficiency of holoprotein, replacement of this loop with various homologous and heterologous loop sequences resulted in folded, cytochrome c-like molecules(6, 11) . Loop A can tolerate a large variation in sequence and structure, including one heterologous loop from an unrelated protein, suggesting that any loop-like sequence would fit into this surface loop and that the specific sequence is not important to protein biosynthesis.
Unlike
other loops, the replacement of the yeast loop D sequence with
sequences that are capable of forming loops in their native protein
does not result in the production of a heme-bound protein, even though
this is a typical -loop found at the surface of the protein (Fig. 1). A low level of apoprotein is present in loop D mutant
yeast strains (Fig. 5) comparable to the levels of wild type
apoprotein found in yeast cells lacking heme lyase(27) .
Minimally, a low level of holoprotein should be produced in these yeast
strains unless apoprotein conversion to holoprotein is blocked or
holoprotein is rapidly degraded. To be undetectable in both the in
vivo functional and structural assays, the mutant holoproteins
would have to be degraded very rapidly to a level below 1-5% (12) of normal cytochrome c. Thus, this is a unique
example of a loop in which replacement of the loop structure with a
homologous loop structure does not restore the overall structure of the
protein. Further, in vivo results suggest that the loop D may
play a role in conversion of apo- to holocytochrome c.
Our in vivo results concur with these in vitro results.
Loop D replacement mutations, where loop D is replaced with homologous
loops from prokaryotic cytochromes c, do not produce
holoprotein and cannot compete with wild type cytochrome c for
mitochondrial import in vivo. In contrast, a carboxyl-terminal
helix deletion mutation, which also does not yield holoprotein, was
able to compete with wild type apocytochrome c for apoprotein
processing. ()Therefore, a mutant apoprotein that contains
the wild type loop D sequence (as well as the rest of the protein) can
compete with wild type holoprotein production, but apoproteins that
lack the wild type loop D sequence are unable to block wild type
cytochrome c bioprocessing.
Sequence variability histograms
show that the loop D sequence,
Asn-Pro-Lys-Lys-Tyr-Ile-Pro-Gly-Thr-Lys-Met-Ala-Phe-Gly-Gly, residues
70-84 in yeast, is highly conserved within the eukaryotic
cytochrome c sequences(36) . This degree of residue
conservation is not observed in the smaller sample of prokaryotic
sequences. In three of the prokaryotic cytochromes c whose
crystal structure has been solved, P. aeruginosa(37) , P. denitrificans(38) , and Rhodospirillum
rubrum(39) , the -loop D secondary structure is
present; however, in these prokaryotic proteins, the amino acid
sequence is not conserved, and this loop is the site of insertions and
deletions, as are other
-loops in the c-type
cytochromes. The sequence conservation of this loop and the results of
the loop replacement experiments support the hypothesis that the
eukaryotic loop D sequence or structure is involved at a step in the
bioprocessing of apocytochrome c, such as mitochondrial
recognition or import, heme lyase recognition or reactivity, heme
binding or folding.
The 3R and 4R oligonucleotides that produced the Pro-76 and Gly-77 mutants were designed by randomizing the first two bases of each codon with all four nucleotides while the third position was synthesized only with G and C. The 8R and 9R oligonucleotides were designed by randomizing the first two bases of the codons encoding Thr-78 and Lys-79, and Tyr-74 and Ile-75, respectively, with all four nucleotides while the third position was randomized only with G and T. By randomizing the third codon position with only two nucleotides, a more even distribution of codons for each of the 20 amino acids is maintained, and overwhelming preferences for amino acids encoded by 6 codons are eliminated. G and T were utilized at the third position in the 8R and 9R oligonucleotides to maximize yeast codon usage preference.
What can the sequences of these random mutants tell us about the structure and function of loop D in cytochrome c? First, and most obviously, the exact sequence of these six residues is not essential for biosynthesis of a folded cytochrome c. What else can be learned from these sequences? If the allowed sequence space has been sampled sufficiently, these sequences can suggest amino acid characteristics that are necessary for cytochrome c function. Two indications that sequence space has been adequately sampled are if the wild type sequence is found among the mutants and if sequence combinations are found multiple times. As detailed under ``Results,'' two of the three experiments returned the wild type sequence, and a number of mutant combinations occurred several times in each experiment (Table 2, Table 3, and Table 4). Thus, sequence space has probably been sufficiently sampled to allow us to suggest amino acid preferences that produce a functional cytochrome c, particularly for Pro-76, Gly-77, Thr-78, and Lys-79 mutants.
The strongest amino acid conservation among the random mutants is found at Ile-75, where only hydrophobic residues are found (Table 2). Valine and leucine are strongly favored. This result concurs with the analysis of the sequence data base, where only isoleucine, methionine, and valine are found at this position. Ile-75 is found 98% buried (solvent exposed area (40) divided by the solvent exposed area in an Ala-Xaa-Ala model peptide) in a hydrophobic core that includes Trp-59, Tyr-67, Val-57, and Asn-52 (Fig. 6), implying that hydrophobic packing plays an important role in residue preference at this position.
Figure 6: A stereoview of the interactions of loop D with the rest of cytochrome c. Residues 34-87 from yeast iso-1-cytochrome c are shown. Loop C (residues 40-54) is light gray, and loop D (residues 70-84) is darker gray. The six residues that were randomized are shown and labeled with their side chains in darkest gray. Loop D residues that participate in hydrogen bonds, as described by Louie and Brayer(47) , are shown as light gray dashed lines.
Thr-78 is 100% buried in iso-1-cytochrome c, and its side chain participates in two hydrogen bonds, one with the heme group and one with the backbone of Met-80 (Fig. 6). This residue can be replaced with alanine, glycine, serine, valine, threonine, isoleucine, and asparagine (Table 6). When a hydrophobic residue is found at position 78, it is not always compensated for by a hydrophilic residue in position 79 (see 8R-9, 10, 12, 14, 16, 17, 19, 28, and 42 in Table 4). These data suggest that neither packing nor hydrogen bonding is essential for biosynthesis, folding, or function. Analysis of the in vitro properties of these mutants will be performed to study the role of packing and hydrogen bonding interactions on protein stability.
Tyr-74 is 77% buried in the structure of cytochrome c. Its hydroxyl group participates in a hydrogen bond with the side chain of Asn-63 (Fig. 6). In the random mutants, tryptophan, glycine, arginine, serine, and valine are found more than once (Table 6). Other amino acids, including the native tyrosine, occur once or twice. The side chain of Lys-79 participates in a hydrogen bond with the backbone of residue 47, and this residue is 61% exposed in the iso-1-cytochrome c crystal structure. Positively charged residues occur 10 times at this position; however, serine is also found here 10 times, and alanine and glycine are each found here four times. At both of these positions, preferences for both large and small residues are striking.
Pro-76 and Gly-77 are strictly conserved throughout the eukaryotic sequences. This conservation is expected because the psi backbone dihedral angle of Gly-77 is 1.5°, a conformation that is not allowed for residues with larger side chains. Thus, as expected, glycine is commonly found at these two positions, 11 times at residue 76 and 12 times at residue 77. However, arginine is also preferred at this position, as it is found eight and seven times at positions 76 and 77, respectively. Once more, the preference for large, positively charged residues, and small residues at the same position is striking and leads to the suggestion that positively charged residues might play a role in the structure or function of cytochrome c in this region. Such contributions will be more fully analyzed by studying the stability and function of these mutant proteins in vitro.
Why, then, are these residues so strictly
conserved during the evolution of eukaryotic cytochromes c? In vivo analysis of the mutants at residues 71 and 80 shows
that many of these changes have a significant effect on the function of
cytochrome c. Preliminary analysis of several of our random
mutants shows drastic effects on protein function in
vivo, thus suggesting that this region of the protein
is critical to proper functioning of cytochrome c.
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