Biomedical subjects

V K Vinson

Publications and source records attributed to V K Vinson.

5 recordsLinked to original sources

PubMed1999

Profilin is predominantly associated with monomeric actin in Acanthamoeba.

We used biochemical fractionation, immunoassays and microscopy of live and fixed Acanthamoeba to determine how much profilin is bound to its known ligands: actin, membrane PIP(2), Arp2/3 complex and polyproline sequences. Virtually all profilin is soluble after gentle homogenization of cells. During gel filtration of extracts on Sephadex G75, approximately 60% of profilin chromatographs with monomeric actin, 40% is free and none voids with Arp2/3 complex or other large particles. Selective monoclonal antibodies confirm that most of the profilin is bound to actin: 65% in extract immunoadsorption assays and 74-89% by fluorescent antibody staining. Other than monomeric actin, no major profilin ligands are detected in crude extracts. Profilin-II labeled with rhodamine on cysteine at position 58 retains its affinity for actin, PIP(2) and poly-L-proline. When syringe-loaded into live cells, it distributes throughout the cytoplasm, is excluded from membrane-bounded organelles, and concentrates in lamellapodia and sites of endocytosis but not directly on the plasma membrane. Some profilin fluorescence appears punctate, but since no particulate profilin is detected biochemically, these spots may be soluble profilin between organelles that exclude profilin. The distribution of profilin in fixed human A431 cells is similar to that in amoebas. Our results show that the major pool of polymerizable actin monomers is complexed with profilin and spread throughout the cytoplasm.

Acanthamoeba↗

PubMed1998

Interactions of Acanthamoeba profilin with actin and nucleotides bound to actin.

Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity of profilin for actin. The affinity for rabbit skeletal muscle actin is about 4-fold lower. The affinity for both actins is 5-8-fold lower with ADP bound to actin rather than ATP. Pyrenyliodoacetamide labeling of cysteine 374 of muscle actin reduces the affinity for profilin 10-fold. The affinity of profilin for nucleotide-free actin is approximately 3-fold higher than for Mg-ATP-actin and approximately 24-fold higher than for Mg-ADP-actin. As a result, profilin binding reduces the affinity of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. Mg-ATP dissociates 8 times faster from actin-profilin than from actin and binds actin-profilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as fast as actin. Thus, profilin promotes the exchange of ADP for ATP. These properties allow profilin to bind a high proportion of unpolymerized ATP-actin in the cell, suppressing spontaneous nucleation but allowing free barbed ends to elongate at more than 500 subunits/second.

Acanthamoeba↗

PubMed1994

Elucidation of the poly-L-proline binding site in Acanthamoeba profilin I by NMR spectroscopy.

The multifunctional protein profilin is one of the most abundant proteins in the cytoplasm and is thought to regulate actin assembly and the phosphoinositide signaling pathway. Profilin binds to several different ligands including actin, poly-L-proline, and the head groups of polyphosphoinositides. Knowledge of profilin/ligand interactions is important for understanding the physiology of profilin in the cell. As a first step in the characterization of profilin/ligand complexes, we have studied a profilin/poly-L-proline complex in solution using high resolution NMR spectroscopy. Analysis of profilin NOE's and chemical shift data indicates that the protein secondary structure is conserved upon binding to poly-L-proline and that the binding site is located between the N- and C-terminal helices in a region rich in highly conserved aromatic sidechains. This site is adjacent to the proposed binding site for actin. In addition, the rate constant for dissociation of the complex is found to be 1.6 +/- 0.2 x 10(4) s-1.

Acanthamoeba↗

PubMed1993

Secondary structure and topology of Acanthamoeba profilin I as determined by heteronuclear nuclear magnetic resonance spectroscopy.

The protein profilin binds to both actin and the head groups of poly)phosphoinositide)s and may regulate both actin assembly and the phosphoinositide signaling pathway. As a first step in understanding the activity of profilin at the molecular level, we have determined the secondary structure of Acanthamoeba profilin I in solution using multidimensional, heteronuclear NMR spectroscopy. Using a combination of triple-resonance (1H, 13C, 15N) experiments, we obtained virtually complete backbone and side-chain resonance assignments based solely on scalar couplings. 3D and 4D NOESY experiments were then used to determine the secondary structure and global fold of Acanthamoeba profilin I. The central feature of the protein structure is a five-stranded antiparallel beta-sheet flanked by three helices and a short two-stranded antiparallel beta-sheet.

Acanthamoeba↗

PubMed1993

Three-dimensional solution structure of Acanthamoeba profilin-I.

We have determined a medium resolution three-dimensional solution structure of Acanthamoeba profilin-I by multidimensional nuclear magnetic resonance spectroscopy. This 13-kD actin binding protein consists of a five stranded antiparallel beta sheet flanked by NH2- and COOH-terminal helices on one face and by a third helix and a two stranded beta sheet on the other face. Data from actin-profilin cross-linking experiments and the localization of conserved residues between profilins in different phyla indicate that actin binding occurs on the molecular face occupied by the terminal helices. The other face of the molecule contains the residues that differ between Acanthamoeba profilins-I and II and may be important in determining the difference in polyphosphoinositide binding between these isoforms. This suggests that lipids and actin bind to different faces of the molecule.

Acanthamoeba↗