The structure of the 56-residue B1 immunoglobulin-binding domain from streptococcal protein G has been determined in two different crystal forms. The crystal structures were deduced by molecular replacement, based on the structure of the B2 domain (Brookhaven accession code 1PGX). Final R values are 0.174 and 0.198 for orthorhombic and trigonal forms, for diffraction data from 6.0 to 2.07 A and from 6 to 1.92 A, respectively. The orthorhombic crystals have an unusually high packing density for protein crystals, with Vm = 1.66 and a solvent content of 26%. The protein structure is found to be very similar (rms deviation 0.25 A for 56 C alpha's) in the two crystal forms, with an efficiently packed hydrophobic core between a four-stranded beta-sheet and a four-turn alpha-helix. The B1 domain has the same fold and general structure as the B2 domain (rms deviations 0.36 and 0.39 A), despite the six residue differences between them. The crystallographic models differ from NMR-derived models in several local regions, primarily in the loop involving residues 46-51; other significant variations are observed in the helix and in the structure of bound water. The primary crystal contact is the same in both crystal forms, involving both sheet edges to form extended beta-sheets throughout the crystals.

We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4␤؉␣ fold can be transformed into an albumin-binding, 3-␣ fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4␤؉␣ to 3-␣ structure can occur via a single amino acid substitution. On one side of the switch point, the 4␤؉␣ fold is >90% populated (pH 7.2, 20°C). A single mutation switches the conformation to the 3-␣ fold, which is >90% populated (pH 7.2, 20°C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin.evolution ͉ NMR ͉ protein design ͉ protein folding

We have cloned, expressed, and characterized two naturally occurring variations of the IgG-binding domain of streptococcal protein G. The domain is a stable cooperative folding unit of 56 amino acids, which maintains a unique folded structure without disulfide cross-links or tight ligand binding. We have studied the thermodynamics of the unfolding reaction for the two versions of this domain, designated B1 and B2, which differ by six amino acids. They have denaturation temperatures of 87.5 degrees C and 79.4 degrees C, respectively at pH 5.4, as determined by differential scanning calorimetry. Thermodynamic state functions for the unfolding reaction (delta G, delta H, delta S, and delta Cp) have been determined and reveal several interesting insights into the behavior of very small proteins. First, though the B1 domain has a heat denaturation point close to 90 degrees C, it is not unusually stable at physiologically relevant temperatures (delta G = 25 kJ/mol at 37 degrees C). This behavior occurs because the stability profile (delta G vs temperature) is flat and shallow due to the small delta S and delta Cp for unfolding. Related to this point is the second observation that small changes in the free energy of unfolding of the B-domain due to mutation or change in solvent conditions lead to large shifts in the heat denaturation temperature. Third, the magnitude and relative contributions of hydrophobic vs nonhydrophobic forces (per amino acid residue) to the total free energy of folding of the B-domain are remarkably typical of other globular proteins of much larger size.

The structure of the complex supports a unimolecular mechanism for prosubtilisin cleavage, involving a 25 A rearrangement of the SBT N terminus in a late folding step. A mechanism of folding catalysis in which the two helices and their connecting beta strand form a prosegment-stabilized folding nucleus is proposed. While this putative nucleus is stabilized by prosegment binding, the N-terminal and C-terminal subdomains of SBT could fold by propagation.

SummaryAn increasing number of proteins demonstrate the ability to switch between very different fold topologies, expanding their functional utility through new binding interactions. Recent examples of fold switching from naturally occurring and designed systems have a number of common features: (i) The structural transitions require states with diminished stability; (ii) Switching involves flexible regions in one conformer or the other; (iii) A new binding surface is revealed in the alternate fold that can lead to both stabilization of the alternative state and expansion of biological function. Fold switching provides insight into how new folds evolve, but also indicates that an amino acid sequence has more information content than previously thought. A polypeptide chain can encode a stable fold while simultaneously hiding latent propensities for alternative states with novel functions.