The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations 1,2 . Even though some unfolded and partially folded proteins are now known to possess biological function 3 or to be involved in aggregation phenomena associated with disease states 1,4 , experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR 6 pulse-labelling experiments that involve rapid in situ protein refolding 7,8 . These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.Experimental advances in nuclear magnetic resonance (NMR) spectroscopy have led to the characterization of a diverse range of unfolded states of proteins 9 . In many cases the presence of residual structure has been shown 10-13 , but with some significant exceptions 14 the poorly resolved spectra of the unfolded state, arising from conformational exchange and dynamic averaging, have generally hampered structural analysis by NMR. We report here the use of an NMR technique that circumvents some of these problems by transferring Three methodologies are combined in this 'pulse-labelling' experiment ( Fig. 1). (1) Photo-CIDNP (chemically induced dynamic nuclear polarization) 6,22 , a technique for enhancing the NMR signals ('hyperpolarization') of solvent-accessible tryptophan, tyrosine and histidine side chains by means of a laser-induced reaction of the protein with a flavin photosensitizer. (2) Rapid homogeneous mixing of solutions in the NMR sample tube to trigger the folding of a denatured protein on a timescale faster than nuclear spin-lattice relaxation ( Supplementary Fig. 1) 7,8 . To these two techniques we add here, for the first time, (3) transfer of nuclear magnetization via nuclear Overhauser effects (NOEs) from the hyperpolarized side chain protons to neighbouring atoms before the refolding step. As a result, inter-residue contacts in unfolded conformations can be detected in the well-resolved NMR spectrum of the refolded native state.1 H photo-CIDNP measurements were initially performed on the native and denatured states of TC5b. The photo-CIDNP spectrum of native TC5b (Fig. 2b) is considerably simpler than the conventional NMR spectrum (Fig. 2a), because only t...

Employing chemical shift melts and hydrogen/deuterium exchange NMR techniques, we have determined the stabilization of the Trp-cage miniprotein due to multiple alanine insertions within the N-terminal α-helix. Alanine is shown to be uniquely helix-stabilizing and this stabilization is reflected in the global fold stability of the Trp-cage. The associated free energy change per alanine can be utilized to calculate the alanine propagation value. From the Lifson-Roig formulation, the calculated value (w Ala = 1.6) is comparable to those obtained for short, solubilized, alanine-rich helices and is much larger than the values obtained by prior host-guest techniques or in Nterminally templated helices and peptides bearing long contiguous strings of alanines with no capping or solubilizing units present.

To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior. A hexameric oligomer of the cyclic peptide is found in both crystal forms and indicates that under appropriate conditions, this minimized system may also serve as a model for protein-protein interactions.protein-protein interaction | folding simulation target | protein cyclization | peptide oligomerization | H-bonding motifs T rp-cage miniproteins (at 18-20 residue length) are among the smallest peptides to fold into a stable protein-like structure (1, 2); as a result, the Trp-cage fold has emerged as a protein folding paradigm (3, 4). Trp-cage species have been extensively studied experimentally (4-11) and computationally (12-19), but the conclusions regarding the folding process and results drawn from these studies are not in accord at the level that should be attainable for such a small system.The structural model used for testing computational folding methods has, in almost all cases, been that derived by NMR for the initially reported Trp-cage (TC5b), which displays a rather low fold stability, T m ¼ 43°C (1). Additional Trp-cage structures based on NMR measurements are now available (2,6,10,(20)(21)(22). All of these appear to possess a common fold topology that is supported by an extensive set of structuring-induced shifts (CSDs, chemical shift deviations from random coil norms) that are largely the result of ring current effects. The uniformity of these CSDs suggests a well-defined common structure. However, other spectroscopic measurements of Trp-cage molecules have been interpreted as being consistent with a folded structure undergoing a greater degree of dynamic fluctuation (7,8). In addition, "folded" structures from molecular dynamics simulations, with a few notable exceptions (18,19), fail to predict the ring current effects observed by NMR (2). The failure of numerous calculations to reproduce the NMR structures and contrary conclusions from other experimental methods is problematic and points to outstanding issues concerning the continuing* use of a Trp-cage as a benchmark for molecular dynamics folding simulations. Further structural studies of Trp-cage species are required to validate the target and to ascertain the key interatomic interactions leading to the folded state.Herein we report X-ray structure determinations for two crystal forms of a cyclized Trp-cage (23), cyclo-TC1 (-GDAYAQW-LADGGPSSGRPPPSG-). Raising additional questions about the oligomeric state of the molec...

• Amino acid residues comprising B-cell epitopes recognized by neutralizing antifactor VIII antibodies (inhibitors) have been identified. • Amino acids contributing significant antigen-antibody binding avidity are candidates for mutagenesis in the design of less antigenic proteins.Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigenantibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients. (Blood. 2014;123(17):2732-2739 IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is a serious complication that may be encountered when FVIII replacement therapy is administered to patients with hemophilia A (HA). It affects 25% to 30% of the treated HA population, with a peak occurrence after ;14 FVIII infusions.1-3 Autoimmune responses to FVIII can also occur, 4 and although this happens only rarely, the resulting bleeding phenotype can be severe. Inhibitors can be difficult and extremely expensive to manage clinically. Interestingly, porcine FVIII has been used effectively in the clinic as a "bypass" therapy; that is, a therapeutic protein that can evade neutralization by anti-FVIII antibodies in many allo-and autoimmune inhibitor patients. [5][6][7] However, some patients have or could develop antibodies that neutralize porcine FVIII as well, 8 because of antigenic cross-reactivity 9 or because regions in which the porcine sequence differs from the human FVIII sequence stimulate effector T cells, leading to antibody production. Identification of the binding sites (B-cell epitopes) on FVIII that are recognized by inhibitors would allow rational design of novel therapeutic FVIII...

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The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies.