Fast folding of a prototypic polypeptide: The immunoglobulin binding domain of streptococcal protein G

Abstract

The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GBl) of streptococcal protein G has been investigated by quenched-flow deuterium-hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND-NH exchange up to 10-fold higher than that expected for a random coil, occurs within the dead time ( 5 1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half-life of 5.2 ms at 5 "C. Keywords: folding intermediates; protein folding; quenched-flow deuterium-hydrogen exchangeAlthough folding intermediates have attracted considerable attention over the last few years (Kuwajima, 1989;Kim & Baldwin, 1990;Matthews, 1993;Fersht & Dill, 1994;Ptitsyn, 1994), new experimental and theoretical studies have cast doubt on their importance as obligate states on the folding pathway of proteins. In particular, theoretical studies and computer simulations of the folding of simplified models for proteins has led to the proposal of a 3-stage random search mechanism of folding, involving the initial rapid collapse from a random coil state to a random semicompact globule, followed by a rate-determining search for one of the many possible transition states from which the chain rapidly folds to the native state (Camacho & Thirumalai, 1993; Chan & Dill, 1994;Sali et al., 1994). The results of this theoretical study further stimulate the debate (Baldwin, 1994;Creighton, 1994), initiated by a provocative paper by Sosnick et al. (1994), challenging the widely held assumption that transient, partly folded intermediates are responsible for the rapid folding of proteins. In 2 related studies (Elove et al., 1994; Sosnick et al., 1994), it was shown that for cytochrome cat least, folding can occur extremely rapidly if trapped, incorrectly ligated heme intermediates are avoided. Indeed, the authors demonstrated that the experimental conditions could be adjusted such that up to 50-70% of the protein can attain the correct heme ligation directly and thus fold rapidly to the native state.These results reopen the debate of whether slow processes observed in folding reactions are of any significance kinetically for native protein folding or are simply the result of species trapped by optional barriers created by conditions specific to the folding solution. Such misorganization of the polypeptide chain may be connected to particular chemical requirements for the folding of the protein under investigation. These include side-chain protonation states, sulfhydryl chemistry, cis-trans proline isomerizations, and the presence of prosthetic groups or metal binding sites. To date, all experimental stu...

The NMR Solution Structure of the Relaxin (RXFP1) Receptor Lipoprotein Receptor Class A Module and Identification of Key Residues in the N-terminal Region of the Module That Mediate Receptor Activation

“…The NOE constraints were further added to and refined with calculations using XPLOR-NIH (36) (version 2.9.9) using a simulated annealing protocol with molecular dynamics in both torsion and Cartesian space. The final force constants used were 50 kcal mol Ϫ1 for experimental distance constraints, 200 kcal mol Ϫ1 rad Ϫ2 for dihedral angle constraints, and 1.0 kcal mol Ϫ1 for the Ramachandran data base potential of mean force (37,38). The quality of structures was validated using PRO-CHECK-NMR (39), and MOLMOL (40) was used to inspect structures, calculate ring current shifts, and produce figures.…”

Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy

“…Analysis of the data as described by Pace et al (1989) yields a free energy of unfolding in water, AG(H,O), of 4.1 kcal.mol" at pH 2. Thus, the GB1 domain is approximately 15% less stable at pH 2 than at pH 4, where NMR of denatured B1 domain of protein G AG ( H20) has a value of 4.8 kcal mol", as determined from a guanidinium chloride unfolding study (Kuszewski et al, 1994). With these data in hand, we proceeded to characterize the unfolded state of GB1 by NMR at pH 2 in 7.4 M urea.…”

“…andrescu et al, 1994;, and the denatured form of a destabilizing mutant of staphylococcal nuclease . To date the dynamic behavior of unfolded or partially unfolded states has only been characterized to a very limited extent Farrow et al, 1995) The B1 immunoglobulin binding domain of streptococcal protein G (GBl) can be described as a prototypic polypeptide for the purposes of studying protein folding (Kuszewski et al, 1994). In particular, it is a small 56-residue domain that is highly thermostable and contains no disulfide bridges, prolines, or prosthetic groups that could result in structural misorganization during the folding process.…”

Core mutants of the immunoglobulin binding domain of streptococcal protein G: Stability and structural integrity

“…The B1 immunoglobulin binding domain of streptococcal protein G (GB1) can be described as a prototypic polypeptide for the purposes of studying protein folding and stability [15,16]. In particular, it is a small 56 residue domain which is highly thermostable and contains no disulfide bridges, prolines or prosthetic groups that could influence its structural organization and stability.…”