ABSTRACT
Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 A in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.
Subject(s)
Adenylate Kinase/chemistry , Alanine/analogs & derivatives , Coumarins/chemistry , Drug Design , Energy Transfer , Fluorescence , Fluorescent Dyes/chemistry , Naphthalenes/chemistry , Acetamides , Adenylate Kinase/genetics , Alanine/chemical synthesis , Alanine/chemistry , Coumarins/chemical synthesis , Escherichia coli/enzymology , Isoquinolines/chemistry , Molecular Structure , Mutagenesis, Site-Directed , Naphthalenes/chemical synthesis , Salicylates/chemistry , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Sulfhydryl Compounds/chemistry , Tryptophan/chemistryABSTRACT
Human serum amyloid P component (hSAP) and human C-reactive protein (hCRP) are normal serum constituents related to the pentraxin family of plasma proteins. hSAP has morphological and immunochemical identity and extensive sequence similarity to the amyloid P (AP) component found in normal tissues and particularly in amyloid deposits. hCRP and its proteolytic products have been previously shown to bind and to interact with various types of human leukocytes. Binding-displacement experiments with 125I-labeled hSAP and hCRP show that both proteins have specific high-affinity binding sites on normal human polymorphonuclear leukocytes (PMN) and each can compete efficiently with the binding of the other. Scatchard analysis of hSAP-displacement curves reveals a heterogeneous population of hSAP-binding sites existing on the PMN cells, among them about 300,000 low-affinity binding sites with Kd < or = 5 x 10(-6) M and about 30,000 high-affinity binding sites with Kd < or = 5 x 10(-8) M. hAP was found to be degraded by enzymes from human neutrophils to yield a mixture of low-molecular-mass peptides, similarly to the case of CRP reported previously. The binding of hSAP can be efficiently inhibited by this peptide mixture. The results suggest that both hCRP and hSAP, together with related peptides, may participate in vivo in an unknown mechanism of regulation of human neutrophils.
