Cytochrome c folding was initiated using a new solution mixer that provides a time window which covers over 90% of the burst phase unresolved by conventional stop-flow measurements. Folding was followed by resonance Raman scattering. Kinetic analysis of the high frequency Raman data indicates that a nascent phase occurs within the mixing dead time of 100 microseconds. A significant fraction of the protein was found to be trapped in a misfolded bis-histidine form during the nascent phase at pH 4.5, thereby preventing the protein from folding rapidly and homogeneously. The nascent phase was followed by a haem-ligand exchange phase that populates the native histidine-methionine coordinated form through a thermodynamically controlled equilibrium.

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Aggregation of a biotherapeutic is of significant concern and judicious process and formulation development is required to minimize aggregate levels in the final product. Aggregation of a protein in solution is driven by intrinsic and extrinsic factors. In this work we have focused on aggregation as an intrinsic property of the molecule. We have studied the sequences and Fab structures of commercial and non-commercial antibody sequences for their vulnerability towards aggregation by using sequence based computational tools to identify potential aggregation-prone motifs or regions. The mAbs in our dataset contain 2 to 8 aggregation-prone motifs per heavy and light chain pair. Some of these motifs are located in variable domains, primarily in CDRs. Most aggregation-prone motifs are rich in β branched aliphatic and aromatic residues. Hydroxyl-containing Ser/Thr residues are also found in several aggregation-prone motifs while charged residues are rare. The motifs found in light chain CDR3 are glutamine (Q)/asparagine (N) rich. These motifs are similar to the reported aggregation promoting regions found in prion and amyloidogenic proteins that are also rich in Q/N, aliphatic and aromatic residues. The implication is that one possible mechanism for aggregation of mAbs may be through formation of cross-β structures and fibrils. Mapping on the available Fab-receptor/ antigen complex structures reveals that these motifs in CDRs might also contribute significantly towards receptor/antigen binding. Our analysis identifies the opportunity and tools for simultaneous optimization of the therapeutic protein sequence for potency and specificity while reducing vulnerability towards aggregation.

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe-protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s 21 , is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6-coordinate and low-spin in both ferric and ferrous forms in the pH range 5.5±10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino-acid sequence comparison, is involved in the formation of the ferric and ferrous 6-coordinate low-spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5-coordinate and high-spin and the ferric form contains a 6-coordinate high-spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure. [3,6]. Despite this considerable divergence at the amino-acid sequence level, the trHbs are predicted to display a globin fold albeit with substantial residue deletions at either N-or C-terminus and in the CD±D region.Sequence comparisons show that the trHbs possess several of the key residues that are required for ligand binding in vertebrate and nonvertebrate Hbs. These include the proximal histidine at position F8 and the distal residue at position E7, which is almost invariably a histidine or glutamine. In vertebrate and nonvertebrate Hbs, the proximal histidine anchors the heme to the polypeptide chain while the distal E7 residue stabilizes oxygen through hydrogen bonding [7,8]. In a single instance, a residue (Leu) not capable of hydrogen bonding was found at the E7 position in M. tuberculosis trHb [5]. Another distal residue, at position B10, which is invariably tyrosine in trHbs, except for that of N. commune with a histidine residue, has been shown to affect ligand stability by further hydrogen bonding in the trHbs from C. eugametos [9] and M. tuberculosis [5]. A similar role has been shown for the B10 tyrosine residue in the Hb of the nematode Ascaris suum [10,11]. It is notable that C. eugametos and P. caudatum trHbs with the same B10 and E7 distal residues have very different O 2 affinities (P 50 , 0.005 mmHg and 0.6 mmHg, for C. eugametos [9] and P. caudatum [12] trHbs, respectively). This s...

Understanding of the chemical nature of the dioxygen moiety of oxyhemoglobin is crucial for elucidation of its physiological function. In the present work, direct Raman spectroscopic observation of both the FeOO 2 and OOO stretching modes unambiguously establishes the vibrational characteristics of the oxygen-bound heme moiety in the hemoglobins of Chlamydomonas eugametos and Synechocystis PCC6803. In addition to providing the resonance Raman assignment of the OOO stretching mode (1136 cm ؊1 for Chlamydomonas, 1133 cm ؊1 for Synechocystis) in an oxyhemoglobin with an iron-porphyrin, this study also reports unusually low frequencies for the FeOO2 stretching modes (554 cm ؊1 ). The effect of strong hydrogen bonding to the bound oxygen is confirmed by changes in the frequency of the FeOO2 stretching mode on mutation of distal residues. These findings suggest an enzymatic function rather than an oxygen transport role for these hemoglobins.

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Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.