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• XPA is an essential protein for the repair of bulky DNA lesions through Nucleotide Excision Repair. • XPA works in complex with RPA to orient the DNA for processing. • A site-specific Cy3 fluorophore was incorporated on XPA and reports on ssDNA binding dynamics. • Detailed methods to generate fluorescent XPA and conditions to generate and maintain stable protein are described. Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido- L -phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies. [ABSTRACT FROM AUTHOR]

• Site-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics. • Bulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA. • Experiments capture the dynamic ssDNA binding states of the DBD-A and DBD-D domains of human RPA. DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido- L- phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions. [ABSTRACT FROM AUTHOR]

• p-azidophenylalanine (pAzF) was incorporated into NF-ĸB for site-specific labeling for smFRET. • p-benzoylphenylalanine (pBpa) was incorporated into NF-ĸB for UV cross-linking mass spectrometry. • Both pAzF and pBpa can be incorporated into NF-ĸB constructs with the 230 residue intrinsically disordered transactivation domain. The transcription factor NF-ĸB is a central mediator of immune and inflammatory responses. To understand the regulation of NF-ĸB, it is important to probe the underlying thermodynamics, kinetics, and conformational dynamics of the NF-ĸB/IĸBα/DNA interaction network. The development of genetic incorporation of non-canonical amino acids (ncAA) has enabled the installation of biophysical probes into proteins with site specificity. Recent single-molecule FRET (smFRET) studies of NF-ĸB with site-specific labeling via ncAA incorporation revealed the conformational dynamics for kinetic control of DNA-binding mediated by IĸBα. Here we report the design and protocols for incorporating the ncAA p-azidophenylalanine (pAzF) into NF-ĸB and site-specific fluorophore labeling with copper-free click chemistry for smFRET. We also expanded the ncAA toolbox of NF-ĸB to include p-benzoylphenylalanine (pBpa) for UV crosslinking mass spectrometry (XL-MS) and incorporated both pAzF and pBpa into the full-length NF-ĸB RelA subunit which includes the intrinsically disordered transactivation domain. [ABSTRACT FROM AUTHOR]

• Monitoring of human SBDS activity using noncanonical amino acid labeling. • Select domains of SBDS can be site-specifically labeled. • Fluorescence anisotropy captures interactions between labeled SBDS and 60S subunit. The Shwachman-Diamond syndrome (SDS) is a rare inherited ribosomopathy that is predominantly caused by mutations in the Shwachman-Bodian-Diamond Syndrome gene (SBDS). SBDS is a ribosomal maturation factor that is essential for the release of eukaryotic translation initiation factor 6 (eIF6) from 60S ribosomal subunits during the late stages of 60S maturation. Release of eIF6 is critical to permit inter-subunit interactions between the 60S and 40S subunits and to form translationally competent 80S monosomes. SBDS has three key domains that are highly flexible and adopt varied conformations in solution. To better understand the domain dynamics of SBDS upon binding to 60S and to assess the effects of SDS-disease specific mutations, we aimed to site-specifically label individual domains of SBDS. Here we detail the generation of a fluorescently labeled SBDS to monitor the dynamics of select domains upon binding to 60S. We describe the incorporation of 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid in human SBDS. Site-specific labeling of SBDS using fluorophore and assessment of 60S binding activity are also described. Such labeling approaches to capture the interactions of individual domains of SBDS with 60S are also applicable to study the dynamics of other multi-domain proteins that interact with the ribosomal subunits. [ABSTRACT FROM AUTHOR]

Protein biogenesis is poorly understood due to the ribosome that perturbs measurement attempted on the ribosome-bound nascent chain (RNC). Investigating nascent chain dynamics may provide invaluable insight into the co-translational processes such as structure formation or interaction with a chaperone [e.g., the bacterial trigger factor (TF)]. In this study, we aim to establish a platform for studying nascent chain dynamics by exploring the local environment near the fluorescent dye on site-specifically labeled RNCs with time-resolved fluorescence anisotropy. To prepare a quantitative model of fluorescence depolarization, we utilized intrinsically disordered protein bound to ribosome, which helped us couple the sub-nanosecond depolarization with the motion of the nascent chain backbone. This was consistent with zinc-finger-domain-containing RNCs, where the extent of sub-nanosecond motion decreased upon the addition of zinc when the fluorophore was in close proximity of the domain. After the characterization of disordered nascent chain dynamics, we investigated the synthesis of a model cytosolic protein, Entner–Doudoroff aldolase, labeled at different sites during various stages of translation. Depending on the stage of translation, the addition of the TF to the nascent chain led to two different responses in the nascent chain dynamics serendipitously, suggesting steric hindrance between the nascent chain and the chaperone as a mechanism for TF dissociation from the ribosome during translation. Overall, our study demonstrates the possible use of site-specific labeling and time-resolved anisotropy to gain insight on chaperone binding event at various stages of translation and hints on TF co-translational mechanism. [ABSTRACT FROM AUTHOR]

Abstract: SH3 is a ubiquitous domain mediating protein–protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for 15N/19F-trifluoromethyl-phenylalanine (15N/19F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide (15N)1H and side-chain 19F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide (15N)1H and side-chain 19F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes. [ABSTRACT FROM AUTHOR]

Human fibroblast growth factor-1 (hFGF1) binding to its receptor and heparin play critical roles in cell proliferation, angiogenesis and wound healing but is also implicated in cancer. Fluorescence imaging is a powerful approach to study such protein interactions, but it is not always obvious if the site chosen will be efficiently labeled, often relying on trial-and-error. To provide a more systematic approach towards an efficient site-specific labeling strategy, we labeled two structurally distinct regions of the protein – the flexible N-terminus and a rigid loop. Several dyes were chosen to cover the visible region and to investigate how the structure of the dye affects the labeling efficiency. Flexibility in either the protein labeling site or the dye structure was found to result in high labeling efficiency, but flexibility in both resulted in a significant decrease in labeling efficiency. Conversely, too much rigidity in both can result in dye-protein interactions that can aggregate the protein. Importantly, site-specifically labeling hFGF1 in these regions maintained biological activity. These results could be applicable to other proteins by considering the flexibility of both the protein labeling site and the dye structure. [Display omitted] • Labeled hFGF1 with a wide range of fluorescent dyes - in both the flexible N-terminal region and in a Rigid β-Turn region. • Cyanine-based dyes efficiently labeled the β-Turn region while rhodamine-based dyes efficiently labeled the N-terminus. • Rigid Rhodamine dye labels on the rigid β-Turn region leads to dye-protein interactions that can aggregate the protein. • HFGF1 labeled in either region maintained biological activity. [ABSTRACT FROM AUTHOR]