In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability.

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.

Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein.

Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.

FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis.

Adaptation to seasonal change is a crucial component of an organism's survival strategy. To monitor seasonal variation, organisms have developed the capacity to measure day length (photoperiodism). Day-length assessment involves the photoperiodic control of flowering in Arabidopsis thaliana, whereby the coincidence of light and high expression of CONSTANS (CO) induces the expression of FLOWERING LOCUS T (FT), leading to flowering in long-day conditions. Although controlling CO expression is clearly a key step in day-length discrimination, the mechanism that generates day-length-dependent CO expression remains unknown. Here we show that the clock-controlled FLAVIN-BINDING, KELCH REPEAT, F-BOX (FKF1) protein has an essential role in generating the diurnal CO peak and that this function is dependent on light. We show that a recombinant FKF1 LIGHT, OXYGEN OR VOLTAGE (LOV) domain binds the chromophore flavin mononucleotide and undergoes light-induced photochemistry, indicating that FKF1 may function as a photoperiodic blue-light receptor. It is likely that the circadian control of FKF1 expression and the light regulation of FKF1 function coincide to control the daytime CO waveform precisely, which in turn is crucial for day-length discrimination by Arabidopsis.

An Improved Method of Predicting Extinction Coefficients for the Determination of Protein Concentration.

Concentration determination is an important method of protein characterization required in the development of protein therapeutics. There are many known methods for determining the concentration of a protein solution, but the easiest to implement in a manufacturing setting is absorption spectroscopy in the ultraviolet region. For typical proteins composed of the standard amino acids, absorption at wavelengths near 280 nm is due to the three amino acid chromophores tryptophan, tyrosine, and phenylalanine in addition to a contribution from disulfide bonds. According to the Beer-Lambert law, absorbance is proportional to concentration and path length, with the proportionality constant being the extinction coefficient. Typically the extinction coefficient of proteins is experimentally determined by measuring a solution absorbance then experimentally determining the concentration, a measurement with some inherent variability depending on the method used. In this study, extinction coefficients were calculated based on the measured absorbance of model compounds of the four amino acid chromophores. These calculated values for an unfolded protein were then compared with an experimental concentration determination based on enzymatic digestion of proteins. The experimentally determined extinction coefficient for the native proteins was consistently found to be 1.05 times the calculated value for the unfolded proteins for a wide range of proteins with good accuracy and precision under well-controlled experimental conditions. The value of 1.05 times the calculated value was termed the predicted extinction coefficient. Statistical analysis shows that the differences between predicted and experimentally determined coefficients are scattered randomly, indicating no systematic bias between the values among the proteins measured. The predicted extinction coefficient was found to be accurate and not subject to the inherent variability of experimental methods. We propose the use of a predicted extinction coefficient for determining the protein concentration of therapeutic proteins starting from early development through the lifecycle of the product.LAY ABSTRACT: Knowing the concentration of a protein in a pharmaceutical solution is important to the drug's development and posology. There are many ways to determine the concentration, but the easiest one to use in a testing lab employs absorption spectroscopy. Absorbance of ultraviolet light by a protein solution is proportional to its concentration and path length; the proportionality constant is the extinction coefficient. The extinction coefficient of a protein therapeutic is usually determined experimentally during early product development and has some inherent method variability. In this study, extinction coefficients of several proteins were calculated based on the measured absorbance of model compounds. These calculated values for an unfolded protein were then compared with experimental concentration determinations based on enzymatic digestion of the proteins. The experimentally determined extinction coefficient for the native protein was 1.05 times the calculated value for the unfolded protein with good accuracy and precision under controlled experimental conditions, so the value of 1.05 times the calculated coefficient was called the predicted extinction coefficient. Comparison of predicted and measured extinction coefficients indicated that the predicted value was very close to the experimentally determined values for the proteins. The predicted extinction coefficient was accurate and removed the variability inherent in experimental methods.

A Spectral Method for Color Quantitation of a Protein Drug Solution.

Color is an important quality attribute for biotherapeutics. In the biotechnology industry, a visual method is most commonly utilized for color characterization of liquid drug protein solutions. The color testing method is used for both batch release and on stability testing for quality control. Using that method, an analyst visually determines the color of the sample by choosing the closest matching European Pharmacopeia reference color solution. The requirement to judge the best match makes it a subjective method. Furthermore, the visual method does not capture data on hue or chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we describe a quantitative method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. Following color industry standards established by International Commission on Illumination, this method converts a protein solution's visible absorption spectra to L*a*b* color space. Color matching is achieved within the L*a*b* color space, a practice that is already widely used in other industries. The work performed here is to facilitate the adoption and transition for the traditional visual assessment method to a quantitative spectral method. We describe here the algorithm used such that the quantitative spectral method correlates with the currently used visual method. In addition, we provide the L*a*b* values for the European Pharmacopeia reference color solutions required for the quantitative method. We have determined these L*a*b* values by gravimetrically preparing and measuring multiple lots of the reference color solutions. We demonstrate that the visual assessment and the quantitative spectral method are comparable using both low- and high-concentration antibody solutions and solutions with varying turbidity. LAY ABSTRACT: In the biotechnology industry, a visual assessment is the most commonly used method for color characterization, batch release, and stability testing of liquid protein drug solutions. Using this method, an analyst visually determines the color of the sample by choosing the closest match to a standard color series. This visual method can be subjective because it requires an analyst to make a judgment of the best match of color of the sample to the standard color series, and it does not capture data on hue and chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we developed a quantitative spectral method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. The details of the spectral quantitative method are described. A comparison between the visual assessment method and spectral quantitative method is presented. This study supports the transition to a quantitative spectral method from the visual assessment method for quality testing of protein solutions.

Validation of a Spectral Method for Quantitative Measurement of Color in Protein Drug Solutions.

A quantitative spectral method has been developed to precisely measure the color of protein solutions. In this method, a spectrophotometer is utilized for capturing the visible absorption spectrum of a protein solution, which can then be converted to color values (L*a*b*) that represent human perception of color in a quantitative three-dimensional space. These quantitative values (L*a*b*) allow for calculating the best match of a sample's color to a European Pharmacopoeia reference color solution. In order to qualify this instrument and assay for use in clinical quality control, a technical assessment was conducted to evaluate the assay suitability and precision. Setting acceptance criteria for this study required development and implementation of a unique statistical method for assessing precision in 3-dimensional space. Different instruments, cuvettes, protein solutions, and analysts were compared in this study. The instrument accuracy, repeatability, and assay precision were determined. The instrument and assay are found suitable for use in assessing color of drug substances and drug products and is comparable to the current European Pharmacopoeia visual assessment method. LAY ABSTRACT: In the biotechnology industry, a visual assessment is the most commonly used method for color characterization, batch release, and stability testing of liquid protein drug solutions. Using this method, an analyst visually determines the color of the sample by choosing the closest match to a standard color series. This visual method can be subjective because it requires an analyst to make a judgment of the best match of color of the sample to the standard color series, and it does not capture data on hue and chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we developed a quantitative spectral method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. In this study, we established a statistical method for assessing precision in 3-dimensional space and demonstrated that the quantitative spectral method is comparable with respect to precision and accuracy to the current European Pharmacopoeia visual assessment method.

Conformational rearrangements in light-harvesting complex II accompanying light-induced chlorophyll a fluorescence quenching.

Light-induced chlorophyll a (Chl a) fluorescence quenching was studied in light-harvesting complex of photosystem II (LHCII). Fluorescence intensity decreased by ca. 20% in the course of 20 min illumination (412 nm, 36 micromol m(-2) s(-1)) and was totally reversible within 30 min dark adaptation. The pronounced quenching was observed only in LHCII in an aggregated form and exclusively in the presence of molecular oxygen. Structural rearrangement of LHCII correlated to the quenching was monitored by measuring changes in UV-Visible light absorption spectra, and by measuring Fourier-transform infrared spectroscopy (FTIR) in the Amide I region of the protein (1600-1700 cm(-1)). The light-induced structural rearrangement of LHCII was interpreted as a partial disaggregation of the complex based on the decrease in the light scattering signal and the characteristic features observed in the FTIR spectra: the relative increase in the intensity of the band at 1653 cm(-1), corresponding to a protein in the alpha-helical structure at the expense of the band centered at 1621 cm(-1), characteristic of aggregated forms. The fact that the light-driven isomerization of the all-trans violaxanthin to the 13-cis form was not observed under the non-oxygenic conditions coincided with the lack of large-scale conformational reorganization of LHCII. The kinetics of this large-scale structural effect does not correspond to the light-induced fluorescence quenching, in contrast to the kinetics of structural changes in LHCII observable at low oxygen concentrations. Photo-conversion of 5% of the pool of all-trans violaxanthin to 9-cis isomer was observed under such conditions. Possible involvement of the violaxanthin isomerization in the process of structural rearrangements and excitation quenching in LHCII is discussed.

Probing antibody internal dynamics with fluorescence anisotropy and molecular dynamics simulations.

The solution dynamics of antibodies are critical to antibody function. We explore the internal solution dynamics of antibody molecules through the combination of time-resolved fluorescence anisotropy experiments on IgG1 with more than two microseconds of all-atom molecular dynamics (MD) simulations in explicit water, an order of magnitude more than in previous simulations. We analyze the correlated motions with a mutual information entropy quantity, and examine state transition rates in a Markov-state model, to give coarse-grained descriptors of the motions. Our MD simulations show that while there are many strongly correlated motions, antibodies are highly flexible, with F(ab) and F(c) domains constantly forming and breaking contacts, both polar and non-polar. We find that salt bridges break and reform, and not always with the same partners. While the MD simulations in explicit water give the right time scales for the motions, the simulated motions are about 3-fold faster than the experiments. Overall, the picture that emerges is that antibodies do not simply fluctuate around a single state of atomic contacts. Rather, in these large molecules, different atoms come in contact during different motions.

A LOV story: the signaling state of the phot1 LOV2 photocycle involves chromophore-triggered protein structure relaxation, as probed by far-UV time-resolved optical rotatory dispersion spectroscopy.

Light-, oxygen-, or voltage-regulated (LOV1 and LOV2) domains bind flavin mononucleotide (FMN) and activate the phototropism photoreceptors phototropin 1 (phot1) and phototropin 2 (phot2) by using energy from absorbed blue light. Upon absorption of blue light, chromophore and protein conformational changes trigger the kinase domain for subsequent autophosphorylation and presumed downstream signal transduction. To date, the light-induced photocycle of the phot1 LOV2 protein is known to involve formation of a triplet flavin mononucleotide (FMN) chromophore followed by the appearance of a FMN adduct within 4 micros [Swartz, T. E., Corchnoy, S. B., Christie, J. M., Lewis, J. W., Szundi, I., Briggs, W. R., and Bogomolni, R. A. (2001) J. Biol. Chem. 276, 36493-36500] before thermal decay back to the dark state. To probe the mechanism by which the blue light information is relayed from the chromophore to the protein, nanosecond time-resolved optical rotatory dispersion (TRORD) spectroscopy, which is a direct probe of global secondary structure, was used to study the phot1 LOV2 protein in the far-UV region. These TRORD experiments reveal a previously unobserved intermediate species (tau approximately 90 micros) that is characterized by a FMN adduct chromophore and partially unfolded secondary structure (LOV390(S2)). This intermediate appears shortly after the formation of the FMN adduct. For LOV2, formation of a long-lived species that is ready to interact with a receptor domain for downstream signaling is much faster by comparison with formation of a similar species in other light-sensing proteins.

Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1.

Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.

Blue-light-activated histidine kinases: two-component sensors in bacteria.

Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis, Brucella abortus, Erythrobacter litoralis, and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

Rhodopsin photointermediates in two-dimensional crystals at physiological temperatures.

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions were studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degrees C. The crystalline environment favored the Meta I(480) photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 degrees C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I(480) decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 degrees C and quenched to 19 degrees C showed that a rapid equilibrium existed on the approximately 1 s time scale, which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII(a)) forms in 2D rhodopsin crystals but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.

Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.

Phototropins (phot1 and phot2) are autophosphorylating serine/threonine kinases that function as photoreceptors for phototropism, light-induced chloroplast movement, and stomatal opening in Arabidopsis. The N-terminal region of phot1 and phot2 contains two specialized PAS domains, designated LOV1 and LOV2, which function as binding sites for the chromophore flavin mononucleotide (FMN). Both LOV1 and LOV2 undergo a self-contained photocycle, which involves the formation of a covalent adduct between the FMN chromophore and a conserved active-site cysteine residue (Cys39). Replacement of Cys39 with alanine abolishes the light-induced photochemical reaction of LOV1 and LOV2. Here we have used the Cys39Ala mutation to investigate the role of LOV1 and LOV2 in regulating phototropin function. Photochemical analysis of a bacterially expressed LOV1 + LOV2 fusion protein indicates that LOV2 functions as the predominant light-sensing domain for phot1. LOV2 also plays a major role in mediating light-dependent autophosphorylation of full-length phot1 expressed in insect cells and transgenic Arabidopsis. Moreover, photochemically active LOV2 alone in full-length phot1 is sufficient to elicit hypocotyl phototropism in transgenic Arabidopsis, whereas photochemically active LOV1 alone is not. Further photochemical and biochemical analyses also indicate that the LOV1 and LOV2 domains of phot2 exhibit distinct roles. The significance for the different roles of the phototropin LOV domains is discussed.

Vibration spectroscopy reveals light-induced chromophore and protein structural changes in the LOV2 domain of the plant blue-light receptor phototropin 1.

Phototropins (phot1 and phot2), the plant blue-light receptors for phototropism, chloroplast movement, and stomatal opening, are flavoproteins that contain two approximately 12 kDa FMN-binding domains, LOV1 and LOV2, at their N-terminus, and a serine/threonine protein kinase domain at their C-terminus. The light-activated LOV2 domain forms a metastable intermediate which has been shown to be a protein-chromophore cysteinyl adduct (Cys39) at C(4a) of FMN. This species thermally relaxes back to the ground state in the dark. We measured the light-minus-dark FTIR difference spectra for the LOV2 domain of oat phot1. These spectra show the disappearance of bands at 1580, 1550, and 1350 cm(-1) that originate from, or are strongly coupled to, the N5=C(4a) stretching vibrations, consistent with the perturbations expected upon C(4a) adduct formation. Assignment of these negative difference FTIR bands to native chromophore vibrations is based on the alignment with resonance Raman bands of FMN. Prominent positive bands include a doublet at 1516 and 1536 cm(-1) and one at 1375 and 1298 cm(-1). Normal-mode vibrational-frequency calculations for both lumiflavin and lumiflavin with a sulfur attached at the C(4a) position agree with many of the positive and negative bands observed in the difference spectra. Both calculated and experimental difference FTIR spectra for deuterium isotope substitutions at exchangeable positions in the flavin chromophore are consistent with the assignment of the above positive bands to vibrational modes involving both the newly formed tetrahedral geometry of C(4a) and the N5-H bond in the long-lived LOV2(S)(390) cysteinyl species.

Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1.

The phototropins are a family of membrane-associated flavoproteins that function as the primary blue light receptors regulating phototropism, chloroplast movements, stomatal opening, and leaf expansion in plants. Phot1, a member of this family, contains two FMN-binding domains, LOV1 and LOV2, within the N-terminal region and a C-terminal serine-threonine protein kinase domain. Light irradiation of oat phot1 LOV2 produces a cysteinyl adduct (Cys-39) at the flavin C(4a) position, which decays thermally back to the dark state. We measured pH and isotope effects on the photocycle. Between pH 3.7 and 9.5, adduct formation showed minimal pH dependence, and adduct decay showed only slight pH dependence, indicating that the pK values of mechanistically relevant groups are outside this range. LOV2 showed a nearly 5-fold slowing of adduct formation in D(2)O relative to H(2)O, indicating that the rate-limiting step involves proton transfer(s). Light-induced changes in the far UV CD spectrum of LOV2 revealed putative protein structural perturbations. The light minus dark CD difference spectrum resembles an inverted alpha-helix spectrum, suggesting that alpha-helicity is reversibly lost upon light irradiation. Decay kinetics for CD spectral changes in the far UV region occur at the same rate as those in the visible region, indicating synchronous relaxation of protein and chromophore structures.

Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.

Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis. Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN). Their C termini contain a serine/threonine protein kinase domain. Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, and Chlamydomonas reinhardtii phot. When expressed in Escherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self-contained photocycle, characterized by fluorescence and absorption changes induced by blue light (T. Sakai, T. Kagawa, M. Kasahara, T.E. Swartz, J.M. Christie, W.R. Briggs, M. Wada, K. Okada [2001] Proc Natl Acad Sci USA 98: 6969-6974; M. Salomon, J.M. Christie, E. Knieb, U. Lempert, W.R. Briggs [2000] Biochemistry 39: 9401-9410). The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness. In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented. Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E. coli. For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies. Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains. The LOV domains of C. reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation. Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.