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Related Concept Videos

Protein Glycosylation01:25

Protein Glycosylation

Glycosylation, the most common post-translational modification for proteins, serves diverse functions. Adding sugars to proteins makes the proteins more resistant to proteolytic digestion. Glycosylated proteins can act as markers and receptors to promote cell-cell adhesion. Additionally, they have many essential quality control functions in the cell, such as correct protein folding and facilitating transport of misfolded proteins to the cytosol, which can be degraded.
Glycosylation occurs in...
Oligosaccharide Assembly01:24

Oligosaccharide Assembly

Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
Proteoglycans01:05

Proteoglycans

Glycans, a class of complex heterogeneous molecules, can be covalently attached to proteins to form glycosylated proteins that regulate various physiological and pathological processes. Glycosylated proteins or glycoproteins comprise N-linked and O-linked oligosaccharides. O-glycosylation is the most common type of protein glycosylation. Here, glycans attach to the oxygen atom of the hydroxyl groups of Serine or Threonine residues. O-linked glycosylation occurs later in protein processing,...
Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...

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Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue
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Protonation and sugar binding to LacY.

Irina N Smirnova1, Vladimir Kasho, H Ronald Kaback

  • 1Department of Physiology and Microbiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-7327, USA.

Proceedings of the National Academy of Sciences of the United States of America
|June 24, 2008
PubMed
Summary

The lactose permease (LacY) maintains sugar binding affinity across physiological pH. However, at high pH (above 9.0), affinity decreases due to faster sugar release, indicating protonation occurs before sugar binding.

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Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
08:53

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Published on: October 2, 2017

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Membrane Transport

Background:

  • Lactose permease (LacY) is a crucial transporter protein.
  • Understanding LacY's pH-dependent behavior is key to its function.
  • Ligand binding affinity is influenced by protein protonation states.

Purpose of the Study:

  • To investigate the impact of bulk-phase pH on lactose permease (LacY) apparent affinity for sugars.
  • To determine the pK(a) value associated with sugar binding.
  • To elucidate the kinetic basis for pH-dependent affinity changes.

Main Methods:

  • Apparent affinity (K(d)(app)) determined using fluorescence-based assays with fluorescent reporters.
  • Measurements conducted across a pH range of 5.5 to 11 for three galactopyranosides.
  • Kinetic analysis of thiodigalactoside binding and direct binding assays with nitrophenylgalactoside.

Main Results:

  • A high pK(a) of approximately 10.5 was observed for all tested galactopyranosides.
  • Decreased sugar affinity at alkaline pH (above 9.0) was attributed to an increased reverse rate (faster dissociation).
  • Sugar binding affinity remained constant between pH 5.5 and 9.0.

Conclusions:

  • LacY exhibits full protonation concerning sugar binding under physiological pH conditions (pH 5.5-9.0).
  • The results support a model where LacY is protonated prior to sugar binding during lactose/H(+) symport.
  • This protonation step is critical for the transport mechanism across the membrane.