HDX-folding competition by NMR None residues whose amide H atoms exchange slowly in the kinetic molten globule MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

A 200 mg/ml sample (14.1 mM) of apo-BLA (denatured and passed through a Pharmacia PD10 column) in 4.5 M GuHCl (H2O) and 15 mM EDTA was diluted 15-fold in 5 mM Tris-2HCl (D2O) at pH 7.5. After five seconds the pH was jumped to 5.5 and the Ca2‡ concentration to 20 mM by mixing with an equal volume of buffer containing 40 mM sodium acetate and 40 mM CaCl2.

Native exchange NMR None log(P) > 5 MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

Rates of solvent exchange of amide hydrogen atoms in the native state of BLA were measured by recording 2D 15N-1H HSQC spectra as a function of time following dissolution of the protein in D2O at pH 6.25.

Native exchange NMR None log(P) < 5 MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

Rates of solvent exchange of amide hydrogen atoms in the native state of BLA were measured by recording 2D 15N-1H HSQC spectra as a function of time following dissolution of the protein in D2O at pH 6.25.

Native exchange in partially folded state by NMR None P > 4 MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

BLA was dissolved in D2O at pH 2.0 for various lengths of time, the solvent exchange was quenched by freezing and the samples were lyophilized. The samples were subsequently analysed under native conditions by re-dissolving in native buffer at pH 5.5 in the presence of Ca2+‡.

Native exchange in partially folded state by NMR None 2.5 < P < 4 MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

BLA was dissolved in D2O at pH 2.0 for various lengths of time, the solvent exchange was quenched by freezing and the samples were lyophilized. The samples were subsequently analysed under native conditions by re-dissolving in native buffer at pH 5.5 in the presence of Ca2+‡.

Native exchange in partially folded state by NMR None 0 < P < 2.5 MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

BLA was dissolved in D2O at pH 2.0 for various lengths of time, the solvent exchange was quenched by freezing and the samples were lyophilized. The samples were subsequently analysed under native conditions by re-dissolving in native buffer at pH 5.5 in the presence of Ca2+‡.

Native exchange NMR None log(P) > 4 MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

A sample of authentic BLA from Sigma (concentration 1.4 mM) for hydrogen exchange was dissolved in 15 mM sodium phosphate buffer in H2O (pH 7.1) containing 20 mM EDTA, and 0.5 M NaCl. After lyophilisation, hydrogen exchange was initiated by dissolving the sample in 100 mM deuterated imidazole buffer (pH 7.1), containing a further 5 mM EDTA. Phase-sensitive COSY spectra 37 were collected on a home-built 500 MHz spectrometer, at 20 °C. A sweep width of 7518 was used in both dimensions. A series of seven COSY spectra was collected with 256 complex t1 increments of 1024 complex points; 32 scans were recorded per t1 increment. The acquisition time was 5.2 hours per spectrum. The data were zero-filled twice to 1 K in the t1 dimension, and zero-filled once to 2 K in the t2 dimension. Resolution enhancement was by trapezoidal and exponential multiplication in the t2 dimension and by trapezoidal multiplication in the t1 dimension.