of Biochemistry, Beckman - PDF document

of Biochemistry, Beckman information about folding pathways exchange, have laboratories and studies more proteins in progress (Baldwin, shown in Figure 1A for T4 polypeptide backbone. Resistance each time the native depends chiefly whether it H-bonded in secondary exchange rates ing intermediates. provides examples obtained from NH protons become protected against change more rapidly in native folding intermediate shows only protection against undergo exchange molten globule model early folding intermediates molten globule intermediate molten globule references therein), find the same protected NH early folding intermediate the equilibrium molten globule Ptitsyn et al. not among the ear- kinetic pathway later intermediates. Their ANS binds only globule species; they observed ANS binding late stage folding reactions several proteins. study showed, however, ANS binding also occurs in burst phase, within milliseconds) (Semisot- nov et of folding. binding occurs later fold- ing intermediates. Molten globule intermediates has long equilibrium folding intermediates (Kuwajima et references therein; Dolgikh equilibrium intermediate the sole various salt be observed probably similar in kinetic folding experiments Ikeguchi et al. early kinetic intermediate same as the equilibrium intermediate. time (ms) unfolded protein) following dilution various times (Bycroft et al., At various times exchange was initiated base catalyzed). folding intermediate formed within stopped-flow mixing time, exchange reflect exchange in N-terminal @-sheet, Ile in helix being analyzed (Baum et general class of folding intermediates that have compact conformations, class of stabilized in by fixed molten globule intermediates. lowered below low salt globule intermediate both intermediates same stability laboratory add unfolded proteins compact intermediates conditions. Truncated deletion specific stabilizing ligands compact, partially struc- side chains influence this compact molten globule intermediates stable relative corresponding unfolded the absence of tertiary in- it possible rapidly, molten of folding. molten globule more stable their unfolded two-state model a more applicable three-state the structures ten globule intermediates not change some folding reactions, transient intermediates Elove et al., isolate and study Interactions stabilizing the apomyoglobin close-packing interactions molten globule intermediate of the assumption unfolding transition responds fitted three-state generate a the effect or both These curves changes only the I transition; the other hand, although mutation affecting I relative does change also affect some of They have as the pre- I reaction, quite different predicted curves reaction. Thus, the fitted three-state kinetic folding in- folding intermediate (solid (Ikeguchi et al., 1986a, redrawn). folding process various concentrations unfolding transition close-packing interactions I transition. Therefore, other in- them in molten globule intermediate. the interactions molten globule intermediate Jorgensen simulated and found a populated intermediate native apoMb comprise a particularly stable subdomain. the individ- lation. The (Waltho et al., Hughson et gate. An upper G peptides can be determined The substitutions in Table reported earlier Hughson et unlike those some interaction must stabilize them a loose hydropho- the hydrophobic introduce a charged chain tend introduce a support the D. Barrick and R.L. Baldwin 0""""""""""' 0.0 2.0 4.0 6.0 8.0 10.0 PH 20,000 15,000 10,000 5,000 0.0 2.0 4.0 6.0 8.0 10.0 PH Fig. 5. A family of curves showing the expected effect of a destabiliz- ing mutation in apoMb that affects either the N Q I reaction (A) or the Io U reaction (B). Successive curves show the effect of decreasing AG" for either reaction by 0.5 series of structure in large fragments staphylococcal nuclease: Effects myoglobin refined Crystallographic refinement of metmyoglobin sperm whale. W.L. (1993). Molecular apomyoglobin in water. stimulating peptide in (solid lines), folding intermediate. interfaces. (Modified tile Barrick.kin,