Protein engineering and stabilization from sequence statistics: variation and covariation analysis.

The concepts of consensus and correlation in multiple sequence alignments (MSAs) have been used in the past to understand and engineer proteins. However, there are multiple ways of acquiring MSA databases and also numerous mathematical metrics that can be applied to calculate each of the parameters. This chapter describes an overall methodology that we have chosen to employ for acquiring and statistically analyzing MSAs.

Simplifying protein expression with ligation-free, traceless and tag-switching plasmids.

Synthetic biology and genome-scale protein work both require rapid and efficient cloning, expression and purification. Tools for co-expression of multiple proteins and production of fusion proteins with purification and solubility tags are often desirable. Here we present a survey of plasmid vectors that provide for some of these features with a focus on tools for rapid cloning and traceless tagging - a setup that facilitates removal of fusion tags post-purification leaving behind no 'scar' on the final construct.

Stabilizing proteins from sequence statistics: the interplay of conservation and correlation in triosephosphate isomerase stability.

Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and high-throughput screens or selections are needed to stabilize proteins using directed evolution.

Triosephosphate isomerase by consensus design: dramatic differences in physical properties and activity of related variants.

Consensus design, the selection of mutations based on the most common amino acid in each position of a multiple sequence alignment, has proven to be an efficient way to engineer stabilized mutants and even to design entire proteins. However, its application has been limited to small motifs or small families of highly related proteins. Also, we have little idea of how information that specifies a protein's properties is distributed between positional effects (consensus) and interactions between positions (correlated occurrences of amino acids).

Contribution of cation-pi interactions to protein stability.

Calculations predict that cation- interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-pi interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-pi interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx).

Design of immunogens that present the crown of the HIV-1 V3 loop in a conformation competent to generate 447-52D-like antibodies.

gp120 is a subunit of the envelope glycoprotein of HIV-1. The third variable loop region of gp120 (V3 loop) contains multiple immunodominant epitopes and is also functionally important for deciding cell-tropism of the virus. 447-52D is a monoclonal antibody that recognizes the conserved tip of the V3 loop in a beta-turn conformation.