The identification and quantification of proteins lags behind DNA sequencing methods in scale, sensitivity and dynamic range. Here we show that sparse amino acid sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescent labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor reductions in each molecule’s fluorescence following consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We demonstrate the method on synthetic and naturally-derived peptide molecules in zeptomole-scale quantities. We also fluorescently label phosphoserines and demonstrate single-molecule, positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should empower studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single molecule sequencing.

CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In addition, efficient “primed” spacer acqui sition and viral degradation (interference) both require the Cascade complex along with the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

Naturally occurring peptides and proteins often use dynamic disulfide bonds to impart defined tertiary/quaternary structures for the formation of binding pockets with uniform size and function. Although peptide synthesis and modification are well established, controlling quaternary structure formation remains a significant challenge. Here, we report the facile incorporation of aryl aldehyde and acyl hydrazide functionalities into peptide oligomers via solid-phase copper-catalysed azide-alkyne cycloaddition (SP-CuAAC) click reactions. When mixed, these complementary functional groups rapidly react in aqueous media at neutral pH to form peptide-peptide intermolecular macrocycles with highly tunable ring sizes. Moreover, sequence-specific figure-of-eight, dumbbell-shaped, zipper-like and multi-loop quaternary structures were formed selectively. Controlling the proportions of reacting peptides with mismatched numbers of complementary reactive groups results in the formation of higher-molecular-weight sequence-defined ladder polymers. This also amplified antimicrobial effectiveness in select cases. This strategy represents a general approach to the creation of complex abiotic peptide quaternary structures.

Bacteria and archaea destroy foreign nucleic acids by mounting an RNA-based CRISPR-Cas adaptive immune response [1][2][3] . In type I CRISPR-Cas systems, the most frequently found type of CRISPR in bacteria and archaea 3,4 , foreign DNAs that trigger efficient immunity can also provoke primed acquisition of protospacers into the CRISPR locus [5][6][7][8][9][10][11][12] . Both interference and primed acquisition require Cascade (CRISPR-associated complex for antiviral defense) and the Cas3 helicase/nuclease. Primed acquisition also requires the Cas1-Cas2 integrase; however, the biophysical mechanisms of how interference and primed acquisition are coordinated have remained elusive. Here, we present single-molecule characterization of the type I-E Thermobifida fusca (Tfu) primed acquisition complex (PAC). TfuCascade rapidly samples non-specific DNA for its target via facilitated one-dimensional (1D) diffusion. An evolutionary-conserved positive patch on the Cse1 subunit increases the target recognition efficiency by promoting this 1D diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex initiates processive translocation via a looped DNA intermediate. Moving Cascade/Cas3 complexes stall and release the DNA loop at protein roadblocks. Cas1-Cas2 samples DNA transiently via 3D collisions, but stably associates with target-bound Cascade. Cas1-Cas2 also remains associated with translocating Cascade/Cas3, forming the PAC. By directly imaging all key subcomplexes involved in target recognition, interference, and primed acquisition, this work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

Single-molecule protein sequencing is regarded as a promising new method in the field of proteomics. It potentially offers orders of magnitude improvements in sensitivitiy and throughput for protein detection when compared to mass spectrometry. However, the development of such a technology faces significant barriers, especially in the need to chemically derivatize specific amino-acid types with unique labels. For example, fluorescent dyes would be suitable for single-molecule microscopy or nanopore-based sequencing. These emerging single-molecule protein-sequencing technologies suggests a need to develop an amino acid side chain-selective modification scheme that could target several side chains of interest. Current work for modifying residues focuses mainly on one or two side chains. The need to label many side chains, as recent computational modeling suggests, is required for high protein, sequencing coverage of the human proteome. Herein, we report our stragety for modifying two model peptides KYDWEC and KDYWE containing the most reactive residues, using highly opitmized mass labels in a sequential and selective fashion both using solution-phase and solid-phase chemistries, respectively. This will serve as a step towards a modification scheme appropriate for single-molecule studies.