The side chain of aspartic acid 69 dictates the folding mechanism of Bacillus subtilis HPr.
Many small, single-domain proteins show equilibrium and kinetic folding mechanisms that appear to be adequately described as two state. The two-state model makes several predictions that can be tested experimentally. First, the conformational stability determined at or extrapolated to a set of reference conditions should be independent of the measurement method (thermal or solvent denaturation or hydrogen exchange). Second, model-independent measures of the cardinal thermodynamic parameters (T(m), DeltaH) as determined from direct calorimetric means should be identical to those determined from the two-state analysis of thermal unfolding data. Third, the ratio of the kinetic folding and unfolding rate constants should be equal to K(eq) determined from an equilibrium measurement under the same conditions. Here, we show that the wild-type HPr protein from Bacillus subtilis does not meet all of these criteria under our standard conditions. However, if we replace the side chain of Asp69, or add moderate concentrations of salt, we find excellent two-state behavior in both equilibrium and kinetic folding. Thus, for this protein and possibly others, very subtle changes in the primary structure or in the solution conditions can dramatically alter the relative stabilities of the native intermediate, and unfolded ensembles can cause an observable change in the nature of the folding mechanism.
Schmittschmitt
Jason P
JP
Department of Medical Biochemistry and Genetics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-1114, USA.
Scholtz
J Martin
JM
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D041961:Deuterium Exchange Measurement; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D012492:Salts / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D012492:Salts; C442131:ScoC protein, Bacillus subtilis; D012997:Solvents; D014157:Transcription Factors; D001224:Aspartic Acid; D012965:Sodium Chloride; D014508:Urea; D019791:Guanidine; D000409:Alanine