The Effects of Protein Charge Patterning on Complex Coacervation

The complex coacervation of proteins with other macromolecules has applications in protein encapsulation and delivery and for determining the function of cellular coacervates. Theoretical or empirical predictions for protein coacervates would enable the design of these coacervates with tunable and predictable structure-function relationships; unfortunately, no such theories exist. To help establish predictive models, the impact of protein-specific parameters on complex coacervation were probed in this study. The complex coacervation of sequence-specific, polypeptide-tagged, GFP variants and a strong synthetic polyelectrolyte was used to evaluate the effects of protein charge patterning on phase behavior. Phase portraits for the protein coacervates demonstrated that charge patterning dictates the protein’s binodal phase boundary. Protein concentrations over 100 mg mL^-1 were achieved in the coacervate phase, with concentrations dependent on the polypeptide sequence. In addition to shifting the binodal phase boundary, polypeptide charge patterning provided entropic advantages over isotropically patterned proteins. Together, these results show that modest changes of only a few amino acids alter the coacervation thermodynamics and can be used to tune the phase behavior of polypeptides or proteins of interest.

The Effects of Protein Charge Patterning on Complex Coacervation

The complex coacervation of proteins with other macromolecules has applications in protein encapsulation and delivery and for determining the function of cellular coacervates. Theoretical or empirical predictions for protein coacervates would enable the design of these coacervates with tunable and predictable structure-function relationships; unfortunately, no such theories exist. To help establish predictive models, the impact of protein-specific parameters on complex coacervation were probed in this study. The complex coacervation of sequence-specific, polypeptide-tagged, GFP variants and a strong synthetic polyelectrolyte was used to evaluate the effects of protein charge patterning on phase behavior. Phase portraits for the protein coacervates demonstrated that charge patterning dictates the protein’s binodal phase boundary. Protein concentrations over 100 mg mL^-1 were achieved in the coacervate phase, with concentrations dependent on the polypeptide sequence. In addition to shifting the binodal phase boundary, polypeptide charge patterning provided entropic advantages over isotropically patterned proteins. Together, these results show that modest changes of only a few amino acids alter the coacervation thermodynamics and can be used to tune the phase behavior of polypeptides or proteins of interest.

Denovo Identification of Cross-linked Peptides via Isotope Coded Linkage Tags

ABSTRACTAn isotope labeled cross-linker (asymmetric d4-DTSSP) was developed to streamline the efforts required for the detection of cross-linked peptides. The cross-linking and mass spectrometry strategy we call Isotope Tagging of Interacting Proteins (iTIP) has improved the specificity of detecting cross-linked peptides and the accurate identification of the interacting peptide sequences via the incorporation of isotopic signatures that are readily observed in the MS/MS spectra. All tryptic peptides derived from the cross-linking reactions of a protein complex are first subjected to ETD-MS2 which results in the facile cleavage of the cross-linker at the disulfide bond and the release of inter-linked polypeptide chains that are detected as a pair of peaks (doublets) in the MS2 spectrum. The constituent peptide halves that are tagged by the heavy/light ends of the cross-linker are easily mass-selected from all other fragment ions, and each polypeptide half is then subjected to CID or HCD-MS3 for identification. The MS3 spectra are subjected to conventional database search strategies available for the sequencing of linear or non-cross-linked peptides. The confident identification of each polypeptide is further assisted by the presence of a stable isotope labeled fragment ions that localizes the cross-linked site on the polypeptide sequence.

Direct Measurement of Light and Heavy Antibody Chains Using Differential Ion Mobility Spectrometry and Middle-Down Mass Spectrometry

The analysis of monoclonal antibodies (mAbs) by a middle-down approach is a growing field that attracts the attention of many researchers and biopharma companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before mass spectrometry (MS) analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light (Lc) and heavy (Hc) chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes ∼ 24 minutes —40-60% faster than regular LC-MS/MS analysis — to acquire fragmentation data using different dissociation methods.

Trigger sequence can influence final morphology in the self-assembly of asymmetric telechelic polymers

We report on a numerical study of polymer network formation of asymmetric biomimetic telechelic polymers with two reactive ends based on a self-assembling collagen, elastin or silk-like polypeptide sequence.

A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.

Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts fromBradyrhizobiumjaponicumUSDA38 and USDA58 by (reduced + carbon monoxide (CO)minusreduced) differential spectroscopy. Database searching showed that onlyfhb,sdgb,gcsandthbgenes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobialglbgenes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobialfhbs andthbs are flanked by a variety of genes whereas several rhizobialsdgbs andgcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra fromB.japonicumUSDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized inB.japonicumUSDA38 and USDA58.

Obtaining complementary polypeptide sequence information from a single precursor ion packet via sequential ion mobility-resolved electron transfer and vibrational activation

Electron transfer, ion mobility, and vibrational activation are combined to obtain temporally-resolved electron transfer dissociation and collision-induced dissociation spectra from a single packet of protonated polypeptide ions.