Putative Interhelix Ion Pairs Involved in the Stability of Myoglobin

Abstract

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable ∆G values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25°C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.Pairs of oppositely charged residues on adjacent helices are predicted to make strong electrostatic interactions in sperm whale Mb 1 (1). These interactions are predicted to be screened by counterions although, if they are present as salt bridges (hydrogen-bonded ion pairs), these are not found to be very sensitive to counterion screening, in peptide helices (2, 3). In either case, if there are any strong pairwise interactions between oppositely charged residues on adjacent helices, they are likely to be important in determining the folding pathway of apoMb. We undertook to measure them, to find out if any changes in stability are indeed caused by pairwise interactions, and to determine how the stability of Mb is affected by counterion screening.Our procedure is to substitute by alanine at least one member of each potential ion pair and to determine the resulting change (∆∆G) in its free energy of unfolding (∆G unf ). If replacing one residue gives a significant change in ∆G unf , then the other residue of the pair is substituted with alanine and ∆∆G is measured to find out if the second mutation causes an equally large change, as expected if there is a pairwise interaction. The test for a pairwise interaction is essential when mutation of one member of a pair gives a large change in stability because other effects, such as steric, hydrophobic, or helix propensity effects, may be responsible for the observed change. To obtain reliable ∆∆G values, the folding transition must be reversible and two-state. We searched for conditions in which the urea-induced unfolding of holoMb satisfies this condition. GdmCl is unsatisfactory for this purpose because high Cl -concentrations stabilize a fold...

The pK a of His-24 in the folding transition state of apomyoglobin

“…In that study, a set of single mutants, each with one basic residue mutated to alanine, consistently increased the stability of the molten globule if the positively charged group was inside the A[B]GH subdomain. Some additional mutants were studied later, and consistent results were found (20). The pK a value of 3.6 assigned to 13 acidic residues (6 Asp, 7 Glu) in the A[B]GH subdomain is the value found from the Linderstrøm-Lang equation with w ϭ 0.05.…”

Towards a “Golden Standard” for computing globin stability: Stability and structure sensitivity of myoglobin mutants

“…These data have been collected from Nishimura et al 29 , Barrick et al 30 , Pinker et al 31 , Luo et al (using the CD-data, not the Trp fluorescence data) 32 , Hargrove et al 33 , by calculating ∆G as -RT ln( 1(K IU K NI ) and computing differences vs. the wild-type, Lin et al 34 , Hughson et al 35 , Ramos et al (using the CD-data) 36 38 , and the low salt (20 mM KPi) data from the thesis by Smith 39 , calculated as ∆G = -RT ln K NU . The average ∆∆G of these 134 data points is ~0.63 kcal/mol, consistent with the general tendency towards ~+1 kcal/mol destabilization from experimental mutation 40 , i.e.…”

Structural Details, Pathways, and Energetics of Unfolding Apomyoglobin

“…Although we know of no experimental data for apomyoglobin, the predicted decrease in stability of , 2 kcal/mol in going from 0 M to 0.1 M NaCl at neutral pH is similar to that observed experimentally on cyanomyoglobin. 42 The inverse Debye length D 21 < 0:316 ffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi ½NaCl p A 21 ; is an appropriate length-scale of the system, and so plotting DG versus D 21 clarifies its salt dependence better than the more common DG versus salt concentration plot. In particular, one does not expect any significant (non-specific) salt effects on folding free energy if the Debye length is considerably larger than the system size, D q R g : For a medium-size protein this is equivalent to monovalent salt concentration being less than , 0.005 M-notice that both lines in Figure 6 do not extrapolate linearly to the origin, but level-off for small D…”