Figure 10

<p><i>Panel a.</i> Far-UV CD spectra of holo-PfRd (native) and different <i>aliphatic</i> residue substitution mutants. <i>Panel b</i>. Far-UV CD spectra of holo-PfRd (native) and different <i>aromatic</i> residue substitution mutants. <i>Panel C</i>. Near-UV aromatic CD spectra of holo-PfRd (native) and different <i>aliphatic</i> residue substitution mutants. <i>Panel D</i>. Near-UV aromatic CD spectra of holo-PfRd (native) and different <i>aromatic</i> residue substitution mutants. <i>Panel E</i>. Near-UV iron-sulphur cluster CD spectra of holo-PfRd (native) and different aliphatic residue substitution mutants. <i>Panel F</i>. Near-UV iron-sulphur cluster CD spectra of holo-PfRd (native) and different aliphatic residue substitution mutants.</p

Figure 9

<p><i>Panel a</i>. The aromatic residue cluster of holo-PfRd. Residues shown are W3 (red), Y10 (blue), Y12 (green), F29 (yellow), W36 (orange), F48 (magenta). <i>Panel b</i>. A schematic representation of the aromatic cluster in holo-PfRd, showing interactions between different aromatic residues. <i>Panel C</i>. The six aromatic residues in the core of holo-PfRD which were mutationally substituted by alanine, shown within the structural context of the polypeptide backbone of the protein. <i>Panel D</i>. The seven aliphatic residues in the periphery of holo-PfRD which were mutationally substituted by alanine, or serine, shown within the structural context of the polypeptide backbone of the protein. <i>Panel E</i>. The sequence of N-terminally 6XHis tagged holo-PfRd. Aromatic residues subjected to substitution mutagenesis are shown in red; Aliphatic residues subjected to substitution mutagenesis are shown in green/blue.</p

2D-1H TOCSY spectra for holo-PfRd (Red), Apo-1 PfRd (blue) and Apo-2 PfRd (green).

<p><i>Panel A</i>. The amide region (finger print region) displaying some residues. Dotted lines indicate the spin systems. <i>Panel b</i>. Aromatic region in the TOCSY spectrum displaying peaks for the side chains of Trp 3, Tyr 10, Tyr 12, Trp 36, and Phe 48. TOCSY cross peaks connecting HZ2, HH2, HZ3 and HE3 are colored red for holo-PfRd and blue for Apo-1. The TOCSY mixing time used for the experiments was 60 msec.</p

Apo-1 PfRd is labile to chemical denaturation, while Apo-2 PfRd resists chemical denaturation.

<p><i>Panel a</i>. Far-UV CD spectra of Apo-1 PfRd at two different concentrations of Gdm.HCl. <i>Panel b</i>. Far-UV CD spectra of Apo-1 PfRd, Apo-2 PfRd and holo-PfRd (native) in 6M Gdm.HCl. <i>Panel c</i>. Fluorescence emission spectra of Apo-2 PfRd at two different concentrations of Gdm.HCl. <i>Panel d</i>. Fluorescence emission spectra of Apo-1 PfRd at two different concentrations of Gdm.HCl.</p

The Key to the Extraordinary Thermal Stability of <i>P. furiosus</i> Holo-Rubredoxin: Iron Binding-Guided Packing of a Core Aromatic Cluster Responsible for High Kinetic Stability of the Native Structure

<div><p><i>Pyrococcus furiosus</i> rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing <i>vis a vis</i> mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status.</p></div

Poor stability of Apo-1 PfRd to denaturation by temperature and 6 M Gdm.HCl.

<p><i>Panel a</i>: Far-UV CD spectra of Apo-1 PfRd at 25°C (blue) and 98°C (black), showing susceptibility to unfolding upon heating. Only the ∼225 nm band is shown. <i>Panel b</i>: Changes in mean residue ellipticity at 222 nm above the temperature of 70°C, shown by Apo-1 PfRd (blue) but not by holo-PfRd (black), as a function of heating. Apo-1 PfRd is lose over half of its CD signal strength at 222 nm. <i>Panel c</i> : Far-UV CD spectra of Apo-1 PfRd in the absence (blue) and presence (black) of 6 M Gdm.HCl. The spectra establish that Apo-1 PfRd loses structure completely (to a greater degree than is achieved by heating) in the presence of the denaturant. <i>Panel d</i>: Emission spectrum of Apo-1 PfRd (blue) shows a profound red shift in the presence of 6 M Gdm.HCl (black), from 335 nm to 355 nm.</p

Similarity between the secondary and tertiary structural features of holo-PfRd, Apo-1 PfRd and Apo-2 PfRd.

<p><i>Panel a</i>: Far-UV CD spectra of Apo-1 (blue), Apo-2 (red) and holo-PfRd (black). The spectrum has two bands. The 203 nm band owes to contributions from polyproline type II (PP-II) and random coil structures. The 225 nm band owes to contributions from secondary structures and aromatic residues. <i>Panel b</i>: Near-UV CD spectra of Apo-1 (blue), Apo-2 (red) and holo-PfRd (black). All bands owe to aromatic contributions. <i>Panel c</i>: Fluorescence emission spectra of Apo-1 (blue), Apo-2 (red) and holo-PfRd (black). The emission maximum of Apo-1 PfRd can be seen to be red-shifted by 2–3 nm.</p

A schematic figure summarizing key conclusions.

<p>A schematic figure summarizing key conclusions.</p

The effects of high temperature and denaturant (6 M Gdm.HCl) on the structure of (native) holo-PfRd.

<p><i>Panel a</i>: Far-UV CD spectra at 25°C (black) and 98°C (orange). <i>Panel b</i>: Fluorescence emission spectra at 25°C (black) and 98°C (orange). <i>Panel c</i>: Far-UV CD spectra in the absence (black) and presence (orange) of 6 M Gdm.HCl, at room temperature. <i>Panel d</i>: Fluorescence emission spectra in the absence (black) and presence (orange) of 6 M Gdm.HCl, at room temperature.</p

Cold denaturation behaviour of PfRd; its reversibility and the effect of B-ME.

<p><i>Panel A</i>: Cold denaturation in PfRd was inhibited by B-ME added at 25°C before initial heating (black curve, 25°C). <i>Panel B</i> :Reversal of cold denaturation, observed through monitoring of reversal in far-UV CD spectral features (secondary structural content). Black curve shows the spectrum of the native form in the presence of Gdm.HCl and B-ME prior to heating; the red curve shows the spectrum at 98°C after first heating; the green curve shows the spectrum at 25°C after cooling from 98°C.<i>Panel C</i> : Cold denaturation in PfRd reversed by B-ME added at 98°C after first heating (black curve, 98°C). No further cold-denaturation (blue curve) seen; instead cold-denaturation is reversed. <i>Panel D</i> : Cold denaturation in PfRd reversed by B-ME added at 25°C. The first heating-cooling cycle is shown by the black (heating) and red (cooling) curves, showing cold-denaturation. Then B-ME was added. The second heating cycle is shown by the green (heating) and blue (cooling) curves, and B-ME is seen to have abrogated cold-denaturation and returned the protein to original state. <i>Panel E</i> :In contrast to the curves in panel D, where B-ME was added at 25°C, here B-ME was added at 98°C, after the second heating. Again, only after B-ME is added, is the reversal of cold-denatuation seen. <i>Panel F</i> : Cold denaturation in PfRd can be reversed significantly by B-ME when added after the first heating-cooling cycle, but only if the temperature at which it is added is above 65–70°C. The black curve is the spectrum of native PfRd and the red curve is the spectrum of cold-denatured PfRd. All other curves represent PfRd's spectrum after heating to a certain temperature (mentioned in the panel), adding B-ME, and cooling back to room temperature. It is seen that if B-ME is added at temperatures below 70°C, the spectrum does not return to that of native PfRd. Otherwise, the spectrum returns to that of PfRd.</p