Stopped-flow fluorescence analysis of GroEL R231W and GroEL CP376-RW.

<p>Experiments were performed at 25°C. Solid traces indicate changes in tryptophan fluorescence for CP376-RW and dotted traces indicate fluorescence changes for GroEL R231W. <i>A.</i> Changes triggered by addition of 1 mM ATP, <i>B.</i> changes triggered by addition of 1 mM ATP and an equimolar concentration of GroES heptamer. In <i>A.</i>, fits to the raw traces are also shown; in white for CP376-RW and in black for R231W. The kinetic constants derived from the fits are as follows: for CP376-RW; <i>k</i>₁ = 145.8±13.2 s⁻¹, Amp₁ = 0.086±0.004, <i>k</i>₂ = 14.2±2.0 s⁻¹, Amp₂ = 0.017±0.002. For R231W; <i>k</i>₁ = 116.4±7.9 s⁻¹, Amp₁ = 0.076±0.003, <i>k</i>₂ = 2.1±0.15 s⁻¹, Amp₂ = −0.037±0.0008. Values are shown as mean ± standard errors. Negative values for amplitude denote phases with increases in fluorescence. <i>C.</i> The changes in the rate constant of Phase B in GroEL CP376-RW as a function of the ATP concentration. Kinetic traces were measured under conditions identical to that for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026462#pone-0026462-g005" target="_blank">Figure 5A</a> and varying the concentration of ATP during measurement. The traces were analyzed to obtain the value of <i>k</i> (± standard error) at each ATP concentration. The results of two separate experimental sessions are shown (<i>blue filled circles</i>). For comparison, results of a previous identical experiment performed on GroEL R231W <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026462#pone.0026462-Taniguchi1" target="_blank">[6]</a> is shown in <i>black circles</i>.</p

N- and C- terminal sequences of the circularly permuted GroEL subunits constructed in this study.

<p><u>Underlines</u> and <>\scale 90%\raster="rg1"<>wavy underlines are added to highlight corresponding sequence elements between each CP mutant and wild type (WT). Sequences in <i>italics</i> denote amino acids that are not from the native sequence of GroEL, added as a consequence of the circular permutation protocol. In addition, we found that in CP376, the N-terminal amino acid sequence had been altered, and apparently insertion of an extra alanine residue had occurred (double underlined). We decided to name this mutant CP376 based on the fact that the native GroEL amino acid sequence continues uninterrupted after Val376. GroEL CP376-RW contains an additional Arg to Trp point mutation at the position corresponding to Arg231 in the wild type sequence.</p

Electron Micrographs of GroEL CP376-RW.

<p>Samples were stained with 1% uranyl acetate. The scale bar in each figure indicates 100 nm. Magnification was x 60,000. <i>Wild Type</i> denotes wild type GroEL, <i>R231W</i> indicates GroEL R231W. <i>CPRW</i> denotes samples of GroEL CP376-RW, and +<i>ATPES</i> indicates samples where 0.2 mM ATP and an equimolar concentration of GroES were added in the course of sample preparation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026462#s4" target="_blank">Materials and Methods</a>). Below each panel, representative zoomed images of an end-on view (left) and side view (right) particle for each sample are shown.</p

ATPase activities of wild type and CP GroELs.

<p><i>A.</i> Basal ATPase activities measured at 25°C. <i>Closed circles</i> indicate wild type GroEL, <i>closed triangles</i> indicate CP209. <i>Open triangles</i> indicate CP254 and <i>open squares</i> indicate CP376 ATPase activities. <i>B.</i> ATPase activities in the absence and presence of an equimolar concentration of GroES heptamer. <i>Columns</i> indicate the concentration of inorganic phosphate that was released after a 60 min incubation at 25°C. Addition of GroES is indicated below each column pair (<i>hatched columns</i>). <i>Labels</i> below each column denote the chaperonin measured.</p

CD spectra of wild type and CP GroELs.

<p>Spectra were recorded at 25°C.</p

A summary of our findings regarding the CP376 GroEL mutant.

<p>For simplicity, only one ring of the GroEL 14-mer is shown. In wild type GroEL (<i>upper</i>), the apical domains are arranged about the heptameric ring in an orderly fashion, and through Phase C, act in a coordinated manner to encapsulate and sequester folding intermediates of rhodanese. In CP376 (<i>lower</i>) however, this orderly orientation is disrupted, and both static and dynamic characteristics of encapsulation are affected. The dynamic aspects of apical domain disorder were observed by the disappearance of the Phase C kinetic transition in stopped-flow experiments, and the static consequences were reflected in an incomplete encapsulation of refolding rhodanese molecules that resulted in Proteinase K sensitivity of the football complex, as well as an ability to bind the cochaperonin GroES in the absence of ATP. Electron micrographs, CD spectra, and stopped-flow assays suggested that these results were caused by an increased flexibility of the apical domain as a functional unit, rather than by an unfolding of this domain caused by circular permutation (for example, the tryptophan fluorescence of CP376-RW continued to reflect various conformational changes of the GroEL subunit in a manner analogous to the wild type subunit).</p

Locations of the N- and C-termini in the three circular permutation mutants of the present study.

<p>An X-ray structural model of a single GroEL/GroES subunit pair taken from PDB structure file 1 AON <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026462#pone.0026462-Xu1" target="_blank">[3]</a>, depicting the three locations where the N- and C-termini were relocated in the three CP mutants. The locations are indicated by colored CPK representation of the first (N-terminal end) amino acid of each CP mutant, excepting the starting methionine and any extraneous amino acids that were added as a consequence of the experimental protocol (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026462#s4" target="_blank">Materials and Methods</a>). The <i>green</i> molecule denotes Glu 209, the <i>yellow</i> molecule Val 254, and the <i>magenta</i> molecule Val 376 in wild type GroEL.</p

Relative stability of the GroEL-ADP-GroES complexes formed at 37°C.

<p>Each GroEL sample was incubated for 2 h at 37°C with an equimolar concentration of GroES. After incubation, samples were partitioned by centrifugal concentration (100 kDa cutoff). Samples denoted "Conc." indicate a 24 µg aliquot of each sample recovered from the upper reservoir concentrate of the filtering apparatus. Samples denoted "Filt." represent proteins that were recovered from a 200 µl aliquot of the lower reservoir filtrate of the apparatus. Experiments were performed in the absence (<i>−ATP, A</i>) and presence (<i>+ATP, B</i>) of 0.2 mM ATP. <i>RW</i> indicates GroEL R231W, and <i>CP</i> indicates GroEL CP376-RW samples. The apparent molecular weights of a commercial marker mixture (Dalton VII molecular weight marker, Sigma) are indicated to the left of the figure.</p