The effects of solution conditions on protein collapse were studied by measuring the hydrodynamic radii of two unfolded proteins, ␣-synuclein and acid-denatured ferricytochrome c, in dilute solution and in 1 M glucose. The radius of ␣-synuclein in dilute solution is less than that predicted for a highly denatured state, and adding 1 M glucose causes further collapse. Circular dichroic data show that ␣-synuclein lacks organized structure in both dilute solution and 1 M glucose. On the other hand, the radius of acid-denatured cytochrome c in dilute solution is consistent with that of a highly denatured state, and 1 M glucose induces collapse to the size and structure of native cytochrome c. Taken together, these data show that ␣-synuclein, a natively unfolded protein, is collapsed even in dilute solution, but lacks structure.

Proteins denature at low pH because of intramolecular electrostatic repulsions. The addition of salt partially overcomes this repulsion for some proteins, yielding a collapsed conformation called the A-state. A-states have characteristics expected for the molten globule, a notional kinetic protein folding intermediate. Here we show that the addition of neutral sugars to solutions of acid-denatured equine ferricytochrome c induces formation of the A-state in the absence of added salt. We characterized the structure and stability of the sugar-induced A-state with circular dichroism spectropolarimetry (CD) and NMR-monitored hydrogen-deuterium exchange experiments. We also examined the stability of the sugar-induced A-state as a function of sugar size and concentration. The results are interpreted using several models and we conclude that the stabilizing effect is consistent with increased steric repulsion between the protein and the sugar solutions.

We present a simple and inexpensive method for producing 15N-labeled Saccharomyces cerevisiae iso-1-cytochrome c in Escherichia coli. The labeled protein gives excellent NMR spectra.

In cells, protein-protein interactions occur in an environment that is crowded with other molecules, but in vitro studies are almost exclusively performed in dilute solution. To gain information about the effects of crowding on protein complex formation, we used isothermal titration calorimetry to measure the stoichiometry, the free energy change, and the enthalpy change for the binding of yeast iso-1-ferricytochrome c to yeast ferricytochrome c peroxidase in dilute solution and in solutions crowded with the sugars glucose, sucrose, and stachyose. The stoichiometry is 1:1 under all conditions. The sugars stabilize the complex, but by only 0.1-0.5 kcal.mol(-)(1), and the increased stability is not correlated with the change in enthalpy of complex formation. We then compared the measured stability changes to values obtained from several analyses that are currently used to predict crowding-induced changes in biomolecular equilibria. None of the analyses are completely successful by themselves, and the results suggest that a complete analysis must account for both excluded-volume and chemical interactions.

Analysis of hyperfine NMR chemical shifts, 1 δ hf , especially for protons, is an established method for probing the structure of redox proteins. 2 For cytochrome c, the redox-state shift, ∆δ rdx , is the difference between the chemical shifts (in ppm) for identical nuclei in the paramagnetic low-spin Fe(III) species and the Fe-(II) diamagnetic reference state, δ ox and δ red , respectively (eq 1).∆δ rdx has contributions from δ hf , the shift from the unpaired electron in the Fe(III) form, and δ str , the shift from redox-related changes in structure. 2d δ hf itself is composed of three terms. δ con is the Fermi contact shift from direct delocalization of the unpaired electron spin on the Fe(III) ion to the heme, the directly linked thioether bonds of C14 and C17, and the axial heme ligands, H18 and M80. 3 δ lc , which does not contribute in the absence of contact shifts, arises from the electron-nuclear dipolar interaction between the nucleus of an atom and the delocalized electron spin in its molecular orbitals. δ mc arises from the electron-nuclear dipolar interaction between a nucleus and the unpaired electron on the Fe(III) ion, which has an anisotropic paramagnetic susceptibility. Analysis of >400 1 H redox shifts for yeast cytochrome c shows that δ mc dominates ∆δ rdx for protons. 2e δ mc also dominates 13 C redox shifts since there is excellent agreement between ∆δ rdx and δ mc for a more limited number of side-chain carbons of horse cytochrome c. 4 The amide proton and nitrogen are close in space, and, as shown in Figure 1A, the calculated δ mc values for these nuclei are highly correlated. We have measured ∆δ rdx for backbone 1 H and 15 N nuclei of yeast iso-1-cytochrome c, specifically, the C102T variant. 5 ∆δ rdx and δ mc for 1 H nuclei are in excellent agreement (Figure 1B), a finding consistent with our previous conclusions 2e and confirming that structures of the two redox states of this protein are similar. The 13 1 H redox shifts that deviate from the calculated δ mc shifts by >|0.25| ppm either are heme ligands (C14, C17, M80) with known contributions from δ con , 3 are near these ligands (R13, L15, Q16), or have known contributions from δ str (G29, H39, G41, A43, A51, D60, K79, A81). 2e,6a Analysis of the 15 N data, however, reveals that many 15 N redox shifts show dramatic deviations from the calculated δ mc values (Figure 1C). Unlike 1 H and 13 C nuclei, 15 N nuclei must experience additional contributions. Additional contributions to 15 N redox shifts are also evident for cytochrome b 5 and Rhodobacter